Acellular matrix implant for treatment of cartilage lesions, injuries and defects

ABSTRACT

An acellular matrix implant for treatment of defects and injuries of articular cartilage, bone or osteochondral bone and a method for treatment of injured, damaged, diseased or aged articular cartilage or bone, using the acellular matrix implant implanted into a joint cartilage lesion in situ and a bone-inducing composition implanted into an osteochondral or bone defect. A method for repair and restoration of the injured, damaged, diseased or aged cartilage or bone into its full functionality by implanting the acellular matrix implant between two layers of biologically acceptable sealants and/or the bone-inducing composition into the osteochondral bone or skeletal bone defect. A method for fabrication of the acellular matrix implant of the invention. A method for preparation of bone-inducing composition.

This application is based on and claims priority of the ProvisionalApplication Ser. No. 60/496,971, filed Aug. 20, 2003, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of Invention

The current invention concerns acellular matrix implants andcompositions for treatment of articular cartilage, bone or osteochondraldefects and injuries and a method for treatment of such osteochondraldefects and/or injured, damaged, diseased or aged articular cartilage orbone using an acellular matrix implant implanted into a joint cartilagelesion and/or into the osteochondral defect in situ wherein theosteochondral or bone defect is further implanted with a bone inducingcomposition or a carrier comprising said composition. The acellularmatrix implant of the invention comprises a two or three dimensionalbiodegradable scaffold structure implanted into the joint cartilagelesion typically below or over one, two or several layers, or betweentwo layers of biologically acceptable sealants. The implant and themethod are particularly useful for repair and restoration of function ofthe injured or traumatized articular cartilage, bone or osteochondraldefects of younger individuals. In particular, the invention concerns amethod where the implantation of the acellular matrix implant of theinvention initiates and achieves natural healing of the cartilage byactivation and migration of chondrocytes from a native, surroundingcartilage into the cartilage defect and/or by inducing bone formation bydepositing a bone inducing composition into the osteochondral and/orbone defect in conjunction with the acellular matrix implant or withoutthe implant.

The method further concerns a formation of a new superficial cartilagelayer overgrowing and sealing the lesion in the joint cartilage byapplying a top sealant over the cartilage lesion as well as insulationof the lesion from the cell and blood debris, by applying a bottomsealant. Such formation of the superficial cartilage layer is alsoapplicable to osteochondral cartilage and bone lesions where the bottomsealant is used for sealing and separating the cartilage and bonelesions and the top sealant is used to form the superficial cartilagelayer.

The method for treatment of articular cartilage comprises preparation ofthe acellular implant, preparation of the lesion for implantation ofsaid implant including a step of depositing a bottom sealant at thebottom of the cartilage lesion for sealing the joint cartilage lesionand protecting the implant from effects of blood-borne agents,implanting the implant of the invention into the lesion and depositingthe top sealant over the implant. The method for treatment ofosteochondral defects additionally typically comprises depositing a boneinducing composition or a carrier comprising said composition into thebone lesion wherein said bone lesion is covered by the bottom sealantthereby separating said bone and cartilage lesions. The method fortreatment of bone defects comprises depositing the bone inducingcomposition or a carrier comprising said composition in a bone lesionwhich may optionally be lined with or covered with a bottom or topsealant.

The invention further concerns a method for repair and restoration ofthe injured, damaged, diseased or aged cartilage or bone into its fullfunctionality and for treatment of injured cartilage by implanting theacellular matrix implant into the cartilage lesion between two or morelayers of biologically acceptable sealants and/or depositing the boneinducing composition or a carrier comprising said composition into thebone lesion, covering said bone inducing composition or a carriercomprising said composition with the bottom sealant, depositing theacellular matrix implant into the cartilage lesion and covering saidimplant with the top sealant.

Additionally, the invention concerns a method for fabrication of anacellular implant of the invention for use in treatment of cartilagedefects and for preparation of a bone inducing composition or a carriercomprising said composition for use in treatment of bone orosteochondral defects.

BACKGROUND AND RELATED DISCLOSURES

Damage to the articular cartilage which occurs in active individuals andolder generation adults as a result of either acute or repetitivetraumatic injury or aging is quite common. Such damaged cartilage leadsto pain, affects mobility and results in debilitating disability.

Typical treatment choices, depending on lesion and symptom severity, arethe rest and other conservative treatments, minor arthroscopic surgeryto clean up and smooth the surface of the damaged cartilage area, andother surgical procedures such as microfracture, drilling, and abrasion.All of these may provide symptomatic relief, but the benefit is usuallyonly temporary, especially if the person's pre-injury activity level ismaintained. For example, severe and chronic forms of knee jointcartilage damage can lead to greater deterioration of the jointcartilage and may eventually lead to a total knee joint replacement.Nowadays, approximately 200,000 total knee replacement operations areperformed annually. The artificial joint generally lasts only 10 to 15years and the operation is, therefore, typically not recommended forpeople under the age of fifty.

Osteochondral diseases or injuries, which are a combination lesions ofbone and cartilage, present yet another challenge for a treatment ofwhich need is not being met by the currently available procedures andmethods. For example, treatment of osteochondritis dissecans withautologous chondrocyte transplantation, described in J. Bone and JointSurgery, 85A-Supplement 2: 17-24 (2003), requires multiple surgeries andat least three weeks for cell cultivation and growth.

It would, therefore, be extremely advantageous to have available amethod for in situ treatment of these injuries which would effectivelyrestore the cartilage or bone to its pre-injury state during one surgeryand with minimal time needed for recovery, which treatment would beespecially suitable for younger individuals who are more active and havebetter recovery capabilities.

Attempts to provide means and methods for repair of articular cartilageare disclosed, for example, in U.S. Pat. Nos. 5,723,331; 5,786,217;6,150,163; 6,294,202; 6,322,563 and in the U.S. patent application Ser.No. 09/896,912, filed on Jun. 29, 2001.

U.S. Pat. No. 5,723,331 describes methods and compositions forpreparation of synthetic cartilage for the repair of articular cartilageusing ex vivo proliferated denuded chondrogenic cells seeded ex vivo, inthe wells containing adhesive surface. These cells redifferentiate andbegin to secrete cartilage-specific extracellular matrix therebyproviding an unlimited amount of synthetic cartilage for surgicaldelivery to a site of the articular defect.

U.S. Pat. No. 5,786,217 describes methods for preparing a multi-celllayered synthetic cartilage patch prepared essentially by the samemethod as described in '331 patent except that the denuded cells arenon-differentiated, and culturing these cells for a time necessary forthese cells to differentiate and form a multicell layered syntheticcartilage.

U.S. application Ser. No. 09/896,912, filed on Jun. 29, 2001 concerns amethod for repairing cartilage, meniscus, ligament, tendon, bone, skin,cornea, periodontal tissues, abscesses, resected tumors and ulcers byintroducing into tissue a temperature dependent polymer gel inconjunction with at least one blood component which adheres to thetissue and promotes support for cell proliferation for repairing thetissue.

U.S. patent application Ser. Nos. 10/104,677; 10/625,822; 10/625,245 and10/626,459 filed on Jul. 22, 2003, by inventors, hereby incorporated byreference, disclose neo-cartilage constructs subjected to an algorithmof certain specific conditions suitable for repair of injured or damagedarticular cartilage.

None of the above cited references, however, results in repair andregeneration of cartilage or bone in situ without a need for severalsurgeries.

It is thus a primary objective of this invention to provide a method anda means for treatment of injured or traumatized cartilage, bone orcartilage-bone defects by depositing at least two separate layers ofbiologically acceptable adhesive sealants thereby forming a cavity inthe injured lesion of the cartilage and implanting an acellular implantinto said cavity between these two layers and, additionally, byproviding a bone inducing composition or a carrier comprising saidcomposition containing bone inducing agents and implanting saidcomposition into the bone lesion of the osteochondral defects followedby the implantation of the acellular matrix implant into the cartilagedefect. The method according to the invention results in induction ofchondrocyte activation and migration from the surrounding nativecartilage into the acellular implant's matrix and in the growth of thesuperficial cartilage layer over the implant thereby sealing the lesionand, when used for treatment of osteochondral defects, in migration ofosteoblast into the bone lesion and in healing of the bone defect aswell as defect of the articular cartilage.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY

One aspect of the current invention is an acellular matrix implant fortreatment of defects and injuries of articular cartilage.

Another aspect of the current invention is an acellular matrix implantin combination with a bone inducing composition or a carrier comprisingsaid composition for treatment of osteochondral defects and injuries.

Still another aspect of the current invention is an acellular boneimplant comprising a bone inducing composition or a carrier comprisingsaid composition for implantation into a bone lesion for treatment ofbone defects and injuries.

Yet another aspect of the current invention is a method for fabricationof an acellular matrix implant of the invention.

Still another aspect of the current invention is a method forpreparation of an acellular matrix implant wherein said matrix is asponge, honeycomb, scaffold, thermo-reversible gelation hydrogel (TRGH)or a polymer of an aromatic organic acid.

Yet another aspect of the current invention is a method for treatment ofinjured, damaged, diseased or aged articular cartilage using theacellular matrix implant implanted into a joint cartilage lesion insitu.

Still yet another aspect of the current invention is an acellular matriximplant used in a method where the implantation of the acellular matriximplant of the invention initiates and achieves activation and inductionof migration of chondrocytes from a native surrounding cartilage intothe acellular matrix implant deposited within a cartilage defect.

Still yet another aspect of the current invention is a method fortreatment of osteochondral defects by implanting an acellular matriximplant into the cartilage lesion in conjunction with depositing a boneinducing composition or a carrier comprising said composition into anosteochondral lesion in situ.

Still another aspect of the current invention is a bone inducingcomposition or a carrier comprising said composition containing boneinducing agents such as a demineralized bone powder, calcium phosphate,hydroxyapatite, organoapatite, titanium oxide, poly-L-lactic orpolyglycolic acid or a copolymer thereof or a bone morphogenic proteinused in a method where the deposition of said composition into the bonelesion initiates migration of osteoblast and achieves natural healing ofthe underlying bone.

Still yet another aspect of the current invention is a bone inducingcomposition or a carrier comprising said composition deposited into abone lesion of the osteochondral defect in conjunction with implantationof an acellular matrix implant into the cartilage lesion useful fortreatment of osteochondral defects.

Still yet another aspect of the current invention is a method fortreatment of bone lesions caused by bone injuries or defects saidtreatment accomplished by implanting a bone inducing composition or acarrier comprising said composition into the bone lesion in situ.

Still another aspect of the current invention is a bone inducingcomposition or a carrier comprising said composition containing boneinducing agents such as a demineralized bone powder, calcium phosphate,hydroxyapatite, organoapatite, titanium oxide, poly-L-lactic orpolyglycolic acid or a copolymer thereof or a bone morphogenic proteinalone, in combination, or incorporated into a carrier, such as a matrix,hydrogel, sponge, honeycomb, scaffold or a polymer of an aromaticorganic acid, used in a method where the deposition of said compositioninto the bone lesion initiates migration of osteoblast and achievesnatural healing of the underlying bone.

Still yet another aspect of the current invention is a bone inducingcomposition or a carrier comprising said composition deposited into abone lesion for treatment of a bone defect alone or, where appropriate,in conjunction with implantation of an acellular matrix implant into thecartilage lesion or osteochondral implant useful for treatment ofosteochondral defects.

Yet another aspect of the current invention is a method for treatment ofinjured, damaged, diseased or aged articular cartilage using anacellular matrix implant implanted into a joint cartilage lesion insitu, said method further comprising a formation of a new superficialcartilage layer overgrowing and sealing the lesion in the jointarticular cartilage by applying a top sealant over the lesion andfurther applying a bottom sealant over the bottom of the lesion, saidbottom sealant providing protection of the lesion against a cell andblood debris migration.

Another aspect of the current invention is a method for treatment ofosteochondral defects by depositing a bone inducing composition or acarrier comprising said composition comprising bone inducing agents intoa bone lesion, depositing a bottom sealant over the bone inducingcomposition or a carrier comprising said composition, implanting anacellular matrix implant into the articular lesion and depositing a topsealant over the acellular matrix implant.

Still another aspect of the current invention is an acellular matriximplant for use in treatments of the cartilage or bone lesionscomprising a two or three dimensional biodegradable sponge, honeycomb,hydrogel, scaffold or a polymer of an aromatic organic acid matriximplanted into the joint cartilage lesion between two layers, top andbottom, of biologically acceptable sealants.

Still yet another aspect of the current invention is a method fortreatment of articular cartilage injury comprising steps:

a) preparation of an acellular matrix implant;

b) preparation of a cartilage lesion for implantation of said implant,including a step of depositing a bottom sealant at the bottom of thecartilage lesion for sealing of said lesion and protecting the implantfrom migration of blood-borne agents;

c) implanting the implant into the lesion; and

d) depositing a top sealant over the acellular matrix implant.

Still yet another aspect of the current invention is a method for repairand restoration of damaged, injured, diseased or aged cartilage to afunctional cartilage, said method comprising steps:

a) preparing an acellular matrix implant as a collagenous sponge,collagenous porous scaffold or honeycomb, thermo-reversible gelationhydrogel (TRGH) or a polymer of an aromatic organic acid matrix, whereinsaid sponge, scaffold, polymer of the aromatic organic acid or TRGH arebiodegradable, will disintegrate with time and be metabolically removedfrom the healed lesion and replaced with a hyaline cartilage, saidmatrix optionally comprising matrix remodeling enzymes, such as matrixmetalloproteinases, aggrecanases, cathepsins and/or other biologicallyactive components;

b) introducing a layer of a biologically acceptable bottom sealant intoa cartilage lesion;

c) implanting said implant into said lesion into a cavity formed by thebottom layer of said bottom sealant; and

d) introducing a top layer of a second biologically acceptable topsealant over said implant wherein said top sealant may or may not be thesame as the bottom sealant and wherein a combination of said implant andsaid top sealant results in formation and growth of a superficialcartilage layer sealing the cartilage lesion in situ.

Still another aspect of the current invention is an acellular matriximplant comprising a thermo-reversible gelation hydrogel (TRGH)deposited into a lesion cavity formed above the bottom sealant layer, orinto the cavity between the top and bottom sealant, said TRGH depositedinto said cavity either incorporated into a collagenous sponge orscaffold or as a sol at temperatures between about 5 to about 30° C.,wherein within said cavity and at the body temperature said TRGHconverts from the fluidic sol into a solid gel and in this form, itspresence provides a structural support for migration of chondrocytesfrom a surrounding native cartilage and formation of extracellularmatrix, wherein said TRGH is biodegradable, will disintegrate with timeand be metabolically removed from the lesion and replaced with a hyalinecartilage.

Still yet another aspect of the current invention is a method fortreatment of osteochondral defects, said method comprising steps:

a) preparing a bone inducing composition or a carrier comprising saidcomposition comprising one or several bone inducing agents forimplantation into a bone lesion;

b) preparing an acellular matrix implant for implantation into acartilage lesion as a collagenous sponge, collagenous porous scaffold orhoneycomb or thermo-reversible gelation hydrogel (TRGH) matrix supportwherein said sponge, scaffold or TRGH are biodegradable, willdisintegrate with time and be metabolically removed from the lesion andreplaced with a hyaline cartilage, said matrix optionally comprisingmatrix remodeling enzymes, matrix metalloproteinases, aggrecanases andcathepsins;

c) introducing said bone inducing composition or a carrier comprisingsaid composition into a bone lesion;

d) covering said bone inducing composition or a carrier comprising saidcomposition with a bottom sealant;

e) implanting said acellular matrix implant into said cartilage lesionover the bottom sealant; and

f) introducing a layer of a top sealant over said implant wherein saidtop and bottom sealants may or may not be the same and wherein acombination of said acellular matrix implant and said top sealantresults in formation and growth of a superficial cartilage layer sealingthe cartilage lesion in situ.

Still yet another aspect of the current invention is a bone inducingcomposition or a carrier comprising said composition comprising boneinducing agents for treatment of osteochondral defects further incombination with an acellular matrix implant comprising athermo-reversible gelation hydrogel (TRGH) each deposited separatelyinto a bone or cartilage lesion, wherein said composition provides ameans for rebuilding the bone and migration of osteoblast into the bonelesion and wherein said implant provides a structural support formigration of chondrocytes from a surrounding native cartilage andformation of extracellular matrix.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is an enlarged schematic representation of the cartilage lesionwithin the host cartilage with underlaying uninjured bone, showing abottom sealant deposited at the bottom of the lesion, an acellularmatrix implant deposited over the bottom sealant and covered with a topsealant. FIG. 1B is an enlarged schematic representation of theosteochondral defect showing the articular lesion, bone lesion,emplacement of the bone inducing composition (bone material) or acarrier comprising said composition into the bone lesion, emplacement oftop and bottom sealants and emplacement of the acellular matrix implant.FIG. 1C is an enlarged schematic representation of the bone defectshowing the articular lesion, and combined osteochondral and skeletalbone lesion, emplacement of the bone inducing composition or a carriercomprising said composition into the bone and osteochondral lesion,emplacement of top and bottom sealants and emplacement of the acellularmatrix implant. FIG. 1D is a schematic depiction of creation of defectsA and B at weight bearing site for implantation of an acellular matriximplant or serving as an empty control defect.

FIG. 2A is an image of an acellular matrix implant held in the forceps.The actual size of the sponge is 5 mm in diameter and 1.5 mm ofthickness. FIG. 2B is a longitudinal scheme of a honeycomb structure ofan acellular matrix implant showing a relative localization of collagensponge and porous collagen gel wherein the pore size is between 200 and400 μm.

FIG. 3 shows a micrograph of the two control empty defect sites A and B(4 mm in diameter and 1-1.5 mm in depth) created on the weight-bearingsite of the swine medial femoral condyle.

FIG. 4 is a micrograph of the two defect sites A and B generated on theweight-bearing site of the swine medial femoral condyle, implanted withacellular matrix implants. The defect has 4 mm in diameter and 1-1.5 mmin depth. The implants have 5 mm diameter and 1.5 mm thickness. Eachimplant is sutured using 4 absorbable sutures and two non-absorbablesutures. The defect was lined up with the bottom sealant and the implantwas covered with the top sealant.

FIG. 5 shows arthroscopic evaluation of a magnified empty defect 2 weeksafter defect creation showing the defect to be fully exposed and empty.

FIG. 6 shows arthroscopic evaluation of a magnified defect treated withthe acellular matrix implant 2 weeks after the defect creation. Thesuperficial cartilage layer overgrowing the implant site forms a smoothflat surface over the defect.

FIG. 7 is a graph illustrating a histological grading of the repairtissue.

FIG. 8A shows a histological evaluation (29× magnification) of the emptydefect (D) at a control site (A). FIG. 8B shows a higher (72×)magnification of the defect site (D). The defect is surrounded by thehost cartilage (H) with underlying subchondral bone (SB) area. Fibroustissue (F) formation is seen in both figures at the empty defect site.Fibrovascular pannus (F) is formed at empty defect site as indicated bythe absence of the S-GAG accumulation.

FIG. 9A shows a histological evaluation (29× magnification) of the emptydefect (D) at a control site (B). FIG. 9B shows a higher (72×)magnification of the defect site (D). The defect is surrounded by thehost cartilage (H) with underlying subchondral bone (SB) area. Fibroustissue (F) formation is seen in both FIGS. 9A and 9B at the empty defectsite with slight accumulation of S-GAG accumulation.

FIG. 10A shows a histological evaluation (29× magnification) of theacellular implantation (I) at the implant site (A). FIG. 10B showsacellular implantation at higher (72×) magnification of the implant site(I). The implant site is surrounded by the host cartilage (H) withunderlying subchondral bone (SB) area. Superficial cartilage layer isshown to cover the implant site. In both FIGS. 10A and 10B normal S-GAGaccumulation and formation of hyaline-like cartilage was observed at theimplant site.

FIG. 11A shows a histological evaluation (29× magnification) of theacellular implantation (I) at the implant site (B). FIG. 11B showsacellular implantation at higher (72×) magnification of the implant site(I). The implant site is surrounded by the host cartilage (H) withunderlying subchondral bone (SB) area. Superficial cartilage layer isshown to cover the implant site. In both FIGS. 11A and 11B normal S-GAG(*) accumulation and formation of hyaline-like cartilage was observed atthe implant site.

FIG. 12 illustrates a degradation pattern in vivo of the top sealant 3months after the acellular matrix implantation. The formed superficialcartilage layer was formed over the implant and the sealant waspartially degraded at three months after the implantation. FIG. 12Ashows a surface view of the Safranin-O stained implantation site. FIG.12B shows a side view of the Safranin-O stained implantation site. FIG.12C shows the bottom view of the Safranin-O stained implantation site.Safranin-O staining, seen as reddish color, indicates S-GAGaccumulation.

FIG. 13 shows an example image of a full thickness defect (D) afterharvest created at femoral condyle of mini-pig at 72× magnification.Surrounding host cartilage (H), subchondral bone area (SB) and remainingcalcified cartilage area are also indicated.

DEFINITIONS

As used herein:

“Acellular” means an implant lacking any biologically active cells.

“Acellular matrix implant” or “acellular implant” means a biologicallyacceptable collagenous implant whether in the form of collagenoussponge, collagenous honeycomb, collagenous scaffold or thermo-reversiblegelation hydrogel without any biologically active cells, forming amatrix into which the chondrocytes may migrate.

“Articular cartilage” means a hyaline cartilage of the joints, such asthe knee joint.

“Subchondral” means a structure underlying a joint cartilage.

“Subchondral bone” means a bone of specific composition, typically verydense, but thin layer of bone just below the zone of calcified cartilageand above the cancellous or trabecular bone that forms the bulk of thebone structure of the limb.

“Osteochondral” means combined area of the cartilage and bone where alesion or lesions occur.

“Osteochondral defect” means a lesion which is a composite lesion ofcartilage and underlying bone.

“Bone defect” or “bone lesion” means the defect which is localized underthe subchondral bone region and is thus a defect/lesion in a skeletalbone.

“Osteoblast” means a bone forming cell.

“Chondrocyte” means a nondividing cartilage cell which occupies a lacunawithin the cartilage matrix.

“Support matrix” means biologically acceptable sol-gel or collagenoussponge, scaffold, honeycomb, hydrogel or a polymer of an aromaticorganic acid suitable for receiving activated migrating chondrocytes orosteocytes that provides a structural support for growth andthree-dimensional propagation of chondrocytes and for formulating of newhyaline cartilage or for migration of osteochondrocytes into the bonelesions. The support matrix is prepared from such materials as Type Icollagen, Type II collagen, Type IV collagen, gelatin, agarose,cell-contracted collagen containing proteoglycans, glycosaminoglycans orglycoproteins, polymers of aromatic organic acids, fibronectin, laminin,bioactive peptide growth factors, cytokines, elastin, fibrin, syntheticpolymeric fibers made of poly-acids such as polylactic, polyglycolic orpolyamino acids, polycaprolactones, polyamino acids, polypeptide gel,copolymers thereof and combinations thereof. The gel solution matrix maybe a polymeric thermo-reversible gelling hydrogel. The support matrix ispreferably biocompatible, biodegradable, hydrophilic, non-reactive, hasa neutral charge and is able to have or has a defined structure.

“Mature hyaline cartilage” means cartilage consisting of groups ofisogenous chondrocytes located within lacunae cavities which arescattered throughout an extracellular collagen matrix.

“Sealant” means a biologically acceptable typically rapidly gellingformulation having a specified range of adhesive and cohesiveproperties. Sealant is thus a biologically acceptable rapidly gellingsynthetic compound having adhesive and/or gluing properties, and istypically a hydrogel, such as derivatized polyethylene glycol (PEG), ora protein, such as albumin, which is preferably cross-linked with acollagen compound. The sealant of the invention typically gels and/orbonds rapidly upon contact with tissue, particularly with tissuecontaining collagen.

“Modified sealant” means any suitable sealant for use in the inventionwhich has a polymerization time of at least 30 seconds.

“Bone-inducing composition” or “a carrier comprising said composition”means a composition comprising at least one bone-inducing agent or,preferably, a combination of several agents, typically dissolved in acarrier or incorporated into a matrix similar to the acellular matriximplant.

“Bone-inducing carrier”, “carrier comprising bone-inducing composition”or “bone acellular implant” means any carrier which containsbone-inducing agents and which by itself promotes bone formation or issuitable for depositing said bone-inducing composition comprising atleast one bone-inducing agent or, preferably, a combination of severalagents. Typically, the carrier will be an acellular biodegradable porousmatrix, hydrogel, sponge, honeycomb, scaffold or a polymer of anaromatic organic acid structure having large pores from about 50 toabout 150 μm, which pores encourage migration of osteoblast andinterconnecting small pores of about 0.1 to about 10 μm which promotesupport and encourage formation of bone. The surface of such carriermight be negatively charged encouraging pseudopod attachment ofosteoblasts and subsequent bone formation. One example of the suitablecarrier promoting bone formation is a polymer of an aromatic organicacid with controllable degree of degradation which is sufficiently hardbut has a spongiform structure.

“Bone-inducing agents” means agents which induce, support or promotebone growth and repair of bone defects. Exemplary bone-inducing agentsare calcium phosphate, hydroxyapatite, organoapatite, titanium oxide,demineralized bone powder, poly-L-lactic and polyglycolic acid or acopolymer thereof or a bone morphogenic protein, among others.

“Bottom sealant” or “first sealant” means a biologically acceptabletissue sealant which is deposited at the bottom of the lesion. In caseof the osteochondral defect, the first sealant is deposited over thebone-inducing composition or a carrier comprising said compositiondeposited into the bone lesion effectively sealing, separating andprotecting the bone lesion from chondrocyte migration as well asprotecting the cartilage lesion from migration of osteocytes.

“Top sealant” or “second sealant” means a biologically acceptablesealant which is deposited above and over the acellular matrix implantimplanted into a lesion and may promote formation of the superficialcartilage layer. The second (top) sealant may or may not be the same asthe first (bottom) sealant and is preferably a cross-linked polyethyleneglycol hydrogel with methyl-collagen.

“De novo” or “de novo formation” means the new production of cells, suchas chondrocytes, fibroblasts, fibrochondrocytes, tenocytes, osteoblastsand stem cells capable of differentiation, or tissues such as cartilageconnective tissue, hyaline cartilage, fibrocartilage, tendon, and bonewithin a support structure, such as multi-layered system, scaffold orcollagen matrix or formation of superficial cartilage layer.

“Superficial cartilage layer” means an outermost layer of cartilage thatforms the layer of squamous-like flattened superficial zone chondrocytescovering the layer of the second sealant and overgrowing the lesion.

“Thermo-reversible” means a compound or composition changing itsphysical properties such as viscosity and consistency, from sol to gel,depending on the temperature. The thermo-reversible composition istypically completely in a sol (liquid) state at between about 5 and 15°C. and in a gel (solid) state at about 25-30° C. and above. The gel/solstate in between shows a lesser or higher degree of viscosity anddepends on the temperature. When the temperature is higher than 15° C.,the sol begins to change into gel and with the temperature closer to30-37° the sol becomes more and more solidified as gel. At lowertemperatures, typically lower than 15° C., the sol has more liquidconsistency.

“TRGH” means thermo-reversible gelation hydrogel material in which thesol-gel transition occurs on the opposite temperature cycle of agar andgelatin gels. Consequently, the viscous fluidic phase is in a sol stageand the solid phase is in a gel stage. TRGH has very quick sol-geltransformation which requires no cure time and occurs simply as afunction of temperature without hysteresis. The sol-gel transitiontemperature can be set at any temperature in the range from 5° C. to 70°C. by molecular design of thermo-reversible gelation polymer (TGP), ahigh molecular weight polymer of which less than 5 wt % is enough forhydrogel formation.

“Sol-gel solution” means a colloidal suspension which, under certainconditions, transitions from a liquid (sol) to a solid material (gel).The “sol” is a suspension of aqueous collagen that is transitioned, byheat treatment, into a gel.

“GAG” means glycosaminoglycan.

“S-GAG” means sulfated glycosaminoglycan.

“Aggrecanase” means aggrecanase enzyme.

“Cathepsin” means a proteinase or peptidase enzyme.

“MMP” means matrix metalloproteinase, an enzyme associated withcartilage degeneration in an injured or diseased joint.

“DMB” means dimethylene blue used for staining of chondrocytes.

“Superficial zone cartilage” means the flattened outermost layer ofchondrocytes covering the extracellular matrix intermediate zone anddeeper zone of mature articular cartilage in which non-dividing cellsare dispersed.

“Connective tissue” means tissue that protect and support the bodyorgans, and also tissues that hold organs together. Examples of suchtissues include mesenchyme, mucous, connective, reticular, elastic,collagenous, bone, blood, or cartilage tissue such as hyaline cartilage,fibrocartilage, and elastic cartilage.

“Adhesive strength” means a peel bond strength measurement, which can beaccomplished by bonding two plastic tabs with an adhesive formulation.The tabs can be formed by cutting 1×5 cm strips from polystyreneweighing boats. To the surface of the boat are bonded (using commercialcyanoacrylate Superglue), sheets of sausage casing (collagen sheeting,available from butcher supply houses). The sausage casing is hydrated inwater or physiological saline for 20 min to one hour and the adhesive isapplied to a 1×1 cm area at one end of the tab; the adhesive is cured.Then, the free ends of the tab are each bent and attached to the upperand lower grips, respectively, of a tensile testing apparatus and pulledat 10 mm/min strain rate, recording the force in Newtons to peel. Aconstant force trace allows estimation of N/m, or force per width of thestrip. A minimum force per width of 10 N/m is desired; 100 N/m or higheris more desirable. Alternatively, the same tab can be bonded (a singletab) over a 1×1 cm area to tissue, either dissected or exposed tissue ina living animal, during surgery. The free end of the tab is then grippedor attached through a perforation to a hook affixed to a hand-heldtensile test device (Omega DFG51-2 digital force gauge; OmegaEngineering, Stamford, Conn.) and pulled upward at approximately 1cm/sec. The maximum force required to detach the tab from the tissue isrecorded. The minimum force desired in such measurements would be 0.1 Nto detach the tab. Forces or 0.2 to 1 N are more desirable.

“Cohesive strength” means the force required to achieve tensile failureand is measured using a tensile test apparatus. The glue or adhesive canbe cured in a “dog-bone”-shaped mold. The wide ends of the formed solidadhesive can then be affixed, using cyanoacrylate (Superglue) to plastictabs, and gripped in the test apparatus. Force at extensional failureshould be at least 0.2 MPa (2 N/cm²) but preferably 0.8 to 1 MPa orhigher.

“Lap shear measurements” means a test of bonding strength, in which thesealant formulation is applied to overlapping tabs of tissue, cured, andthen the force to pull the tabs apart is measured. The test reflectsadhesive and cohesive bonding; strong adhesives will exhibit values of0.5 up to 4-6 N/cm² of overlap area.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on findings that when a biodegradable acellularmatrix implant, such as a collagenous sponge matrix, collagenousscaffold matrix or thermo-reversible gelation hydrogel matrix implant,is deposited into a lesion of injured, traumatized, aged or diseasedcartilage or, in conjunction with a bone-inducing composition or acarrier comprising said composition comprising bone activating agents,into an osteochondral or bone defect, within time, this acellular matriximplant activates mature but non-dividing chondrocytes present in thesurrounding native cartilage, induces them to migrate to a site of thearticular cartilage defect and generates a new extracellular matrixultimately resulting in formation of a healthy hyaline cartilage and/or,in case of the bone or osteochondral defect, it induces migration ofosteoblast cells from surrounding healthy bone or subchondral bone.Under these circumstances, the second, top sealant deposited over theacellular matrix implant will promote in situ formation of superficialcartilage layer over the cartilage lesion containing the implant. Suchsuperficial cartilage layer will be also generated when the top sealantis deposited over the osteochondral defect, which, additionally, willcomprise depositing of the bone-inducing composition or a carriercomprising said composition into the bone lesion and covering saidcomposition with a first, bottom sealant.

The invention thus, in its broadest scope, concerns a method for repairand restoration of damaged, injured, traumatized or aged cartilage orfor repair of bone or osteochondral defects and restoration of both thecartilage and/or bone into their full functionality by implanting,during arthroscopic surgery, an acellular matrix implant and/ordepositing a bone-inducing composition or a carrier comprising saidcomposition into the bone lesion before implanting the acellular matriximplant into the cartilage lesion. The invention further includes amethod for fabrication of said acellular matrix implant, preparation ofsaid bone-inducing composition or a carrier comprising said compositionand a method for de novo formation of a superficial cartilage layer insitu.

Briefly, for treatment of the articular lesions, the invention comprisespreparation of the acellular matrix implant for implanting into a jointcartilage lesion, said implant comprising a collagenous,thermo-reversible gel or an aromatic organic acid polymer support matrixin two or three-dimensions. The acellular matrix implant may containvarious supplements, such as matrix remodeling enzymes,metalloproteinases (MMP-9, MMP-2, MMP-3), aggrecanases, cathepsins,growth factors, donor's serum, ascorbic acid, insulin-transferrin-sodium(ITS) etc., in concentrations which are known in the art to inducegrowth, differentiation and phenotype stability.

For treatment of osteochondral defects, the invention comprisespreparation of a bone-inducing composition or a carrier comprising saidcomposition comprised of bone-inducing agents, such as demineralizedbone powder, calcium phosphate, hydroxyapatite, organoapatite, titaniumoxide, poly-L-lactic and polyglycolic acid or a copolymer thereof, aloneor in combination, or a bone morphogenic protein, depositing saidcomposition into the bone lesion and covering said bone-inducingcomposition or a carrier comprising said composition with the firstbottom sealant followed by depositing said acellular matrix implant intothe cartilage lesion and covering said implant with the second, topsealant.

For treatment of bone defects, the invention comprises preparation of abone-inducing composition or a carrier comprising said compositioncomprised of bone-inducing agents, such as demineralized bone powder,calcium phosphate, hydroxyapatite, organoapatite, titanium oxide,poly-L-lactic and polyglycolic acid or a copolymer thereof, alone or incombination, or a bone morphogenic protein in amounts needed to fill thebone lesion, and depositing said composition into the bone lesion. Saidlesion may optionally be covered with the bottom or top sealant.Typically, the bottom sealant is not deposited at the bottom of the bonelesion but if needed, it can be.

The acellular matrix implant is implanted into a cartilage lesion cavityformed by at least two layers of adhesive sealants. However, in certaincircumstances, the acellular matrix implant may be also deposited intothe cartilage lesion without either the bottom or top sealant or withoutboth sealants.

When the sealants are used in the method for repair of cartilage, thefirst (bottom) layer of the sealant is deposited at and covers thebottom of the cartilage lesion. Its function is to protect the integrityof said lesion from cell migration and from effects of various blood andtissue debris and metabolites and also to form a bottom of the cavityinto which the acellular matrix implant is deposited. The first layer ofthe sealant may also become a covering layer deposited over thebone-inducing composition or a carrier comprising said compositionplaced into the bone lesion within the subchondral bone or bone area.

Studies of induced defects of the pig's femoral condyle confirmed thatimplantation of a biodegradable acellular matrix implant combined with aimplantation procedure disclosed herein and performed under definedconditions induces activation and promotes chondrocyte migration fromsurrounding native host cartilage resulting in formation ofextracellular matrix (ECM) of a regenerated hyaline-like cartilagewithin the lesion at the injured site. Similarly, a deposition of abone-inducing composition or a carrier comprising said compositioncomprising bone-inducing agents into the bone defect promotes naturalhealing of bone by inducing migration of osteoblast into said bonelesion and, combined with the acellular matrix implant as describedabove, leads to healing and reconstruction of both the bone andcartilage.

The method for using the acellular matrix implant for generation of thehyaline cartilage is particularly suitable for treatment of lesions inyounger patients with focused lesions where the cartilage has notdeveloped an incipient osteoarthritic conditions, that is in patientswho would typically be treated with microfracture or with cleaning thearticular cartilage in the joint, such as in, for example, arthroscopicsurgery following a sports injury. Such patients stand a highprobability of restoring a fully functional hyaline cartilage, or incase of osteochondral defects, a fully functional cartilage and bone,without need of and aggravation associated with undergoing additionalone or multiple surgeries.

One advantage of using the above-described method is that the acellularmatrix implant and/or the bone-inducing composition or a carriercomprising such composition is non-immunogenic, can be pre-manufacturedwell before the operation and can be introduced during the firstarthroscopy, when the diagnosis, cleaning and debridement of the lesiontakes place without a need for further biopsy, cell culturing,additional surgeries or treatments to prevent immune reactions.

I. Cartilage, Bone and Properties Thereof

Cartilage and bone, both, are connective tissues providing support inthe body for other soft tissues.

Bone is a hard connective tissue forming a skeleton, consisting ofosteoblast cells embedded in a matrix of mineralized ground substanceand collagen fibers. The collagen fibers are impregnated with a form ofcalcium phosphate similar to hydroxyapatite as well as with substantialquantities of carbonate, citrate, sodium and magnesium. Bone is composedof approximately 75% of inorganic material and 25% of organic material.Bone consists of a dense outer layer of compact substance covered byperiosteum and an inner, loose spongy substance, i.e. bone marrow. Boneemplaced immediately below the cartilage is called subchondral bone andit is a bone of specific composition and structure that is itselfunderlain by the cancellous bone of the limb.

Cartilage is a mature connective tissue covering joints and bones whichis comprised of metabolically active but non-dividing chondrocytes. Thisresults in essential non-existence of spontaneous ability of thecartilage to self-repair following the injury or damage caused by age ordisease.

Cartilage is characterized by its poor vascularity and a firmconsistency, and consists of mature non-dividing chondrocytes (cells),collagen (interstitial matrix of fibers) and a ground proteoglycansubstance (glycoaminoglycans or mucopolysaccharides). Later two arecumulatively known as extracellular matrix.

There are three kinds of cartilage, namely hyaline cartilage, elasticcartilage and fibrocartilage. Hyaline cartilage, found primarily injoints, has a frosted glass appearance with interstitial substancecontaining fine type II collagen fibers obscured by proteoglycan.Elastic cartilage is a cartilage in which, in addition to the collagenfibers and proteoglycan, the cells are surrounded by a capsular matrixfurther surrounded by an interstitial matrix containing elastic fibernetwork. The elastic cartilage is found, for example, in the centralportion of the epiglottis. Fibrocartilage contains Type I collagenfibers and is typically found in transitional tissues between tendons,ligaments or bones and also as a low quality replacement of injuredhyaline cartilage. This invention utilizes properties of acellularmatrix implant combined with certain conditions existing naturally inthe surrounding native cartilage further combined with certain stepsaccording to the method of the invention, to achieve the healing andreplacement of injured cartilage with the healthy and functional hyalinecartilage.

A. Articular Cartilage and Articular Cartilage Defects

The articular cartilage of the joints, such as the knee cartilage, ishyaline cartilage which consists of approximately 5% of chondrocytes(total volume) seeded in approximately 95% extracellular matrix (totalvolume). The extracellular matrix contains a variety of macromolecules,including collagen and glycosaminoglycan (GAG). The structure of thehyaline cartilage matrix allows it to reasonably well absorb shock andwithstand shearing and compression forces. Normal hyaline cartilage hasalso an extremely low coefficient of friction at the articular surface.

Healthy hyaline cartilage has a contiguous consistency without anylesions, tears, cracks, ruptures, holes or shredded surface. Due totrauma, injury, disease such as osteoarthritis, or aging, however, thecontiguous surface of the cartilage is disturbed and the cartilagesurface shows cracks, tears, ruptures, holes or shredded surfaceresulting in cartilage lesions.

The articular cartilage is an unique tissue with no vascular, nerve, orlymphatic supply. The lack of vascular and lymphatic circulation may beone of the reasons why articular cartilage has such a poor, almostnon-existent intrinsic capacity to heal. The mature metabolically activebut non-dividing chondrocytes in their lacunae surrounded byextracellular matrix do not respond to damage signals by generatinghigh-quality hyaline cartilage. After a significant injury, uniquemechanical functions of articular cartilage are never reestablishedspontaneously and never completely because the water-absorption capacityof the type II collagen/proteoglycan network is disturbed. The usualreplacement material for hyaline cartilage, which might developspontaneously in response to the injury of hyaline cartilage and whichreplaces the injured cartilage, is the much weaker and functionallyinferior fibrocartilage.

Defects occurring due to cartilage trauma, injury, disease or aging aretears, cracks, ruptures or holes which are solely located in the jointcartilage. According to the method of the invention, when such defect istreated, the implant is deposited within the lesion, as illustrated inFIG. 1A.

FIG. 1A is a schematic representation of an acellular matrix implantimplantation into the cartilage defect. The scheme shows the lesionimplantation site with acellular matrix implanted therein surrounded byhost cartilage with underlaying undisturbed subchondral bone.Emplacement of the top and bottom sealants are also illustrated.

B. Currently Available Procedures for Repair of Cartilage

A variety of surgical procedures have been developed and used inattempts to repair damaged cartilage. These procedures are performedwith the intent of allowing bone marrow cells to infiltrate the defectand promote its healing. Generally, these procedures are only partly, ifat all, successful. More often than not, these procedures result information of a fibrous cartilage tissue (fibrocartilage) which does filland repair the cartilage lesion but, because it is qualitativelydifferent being made of Type I collagen fibers, it is less durable, lessresilient and generally inferior than the normal articular hyalinecartilage and thus has only a limited ability to withstand shock andshearing forces than does healthy hyaline cartilage. Since alldiarthroid joints, particularly knees joints, are constantly subjectedto relatively large loads and shearing forces, replacement of thehealthy hyaline cartilage with fibrocartilage does not result incomplete tissue repair and functional recovery.

Among the currently available procedures for repair of the articularcartilage injuries are the microfracture technique, the mosaicplastytechnique and autologous chondrocyte implantation (ACI). However, in oneway or another, all these techniques are problematic. The mosaicplastytechnique and ACI, for example, need a biopsy of cartilage from anon-damaged articular cartilage area and subsequent cell culture to growthe number of cells. As a consequence, these techniques require at leasttwo separate surgeries. One system, the Carticel® system additionallyrequires a second surgery site to harvest portion of and, therefore,disrupt the tibial periosteum. While the microfracture technique doesnot require a biopsy of articular cartilage, the resulting tissue whichdevelops is always fibrocartilage.

The method for treatment of injured, traumatized, diseased or agedcartilage according to the current invention obviates the above problemsas it comprises treating the injured, traumatized, diseased or agedcartilage with an acellular matrix implant without need to remove tissueor cells for culturing, said implant prepared by methods described belowand implanted into the cartilage lesion during the debriding surgery, asdescribed below.

C. Osteochondral Area and Osteochondral Defects

Osteochondral area, in this context, means an area where the bone andcartilage connect to each other and where the osteochondral defectsoften develop following the injury.

FIG. 1B is a schematic representation of implantation of an acellularmatrix implant in the osteochondral defect. The scheme shows thecartilage lesion implantation site with the acellular matrix implantedtherein surrounded by host cartilage with underlaying bone lesion in thesubchondral bone. A bone-inducing composition or an acellular implantcarrier comprising said composition is deposited into the bone lesionseparated from the cartilage lesion by the bottom sealant. Emplacementof the top and bottom sealants illustrates separation of the bone lesionfrom the cartilage lesion by the bottom sealant such that each thecartilage lesion and the bone lesion are treated separately usingdifferent means, namely the acellular matrix implant for treatment ofthe cartilage lesion and the bone-inducing composition or the acellularcarrier comprising said composition for treatment of the bone defect.

Osteochondral defects are thus defects that are composites of cartilageand underlying bone. Up-to-date, commonly used treatments forosteochondral defects are surgical excisions, mosaicplasty,osteochondral autogenous grafting, allogenic grafting, bone cementing,deposition of metal or ceramic solid composite materials, porousbiomaterials and, lately, a transplantation of autologous chondrocytes.Regretfully, none of these procedures was found to be successful intreating these defects and safe or comfortable for a patient. Typically,these procedures involve two or more surgical procedures and longperiod, generally at least two to three weeks, of time to culture thetransplantable cells. For example, mosaicplasty requires removal ofcircular pieces of healthy subchondral bone and cartilage to be used astransplantable plugs at a defect site. One obvious problem withmosaicplasty is that the surgeon, in an open surgery, is disruptinghealthy tissue in order to repair the subchondral defect. Clearly, themultiple surgeries and long period of time between them necessarilyextend a time of recovery to fully functional joint and often resultonly in partial functional restoration as both the bone and cartilagedefects are filled with the fibrocartilage instead of the bone andhyaline cartilage.

One example of the osteochondral defect which is common and verydifficult to treat is osteochodritis dissecans. Osteochodritis dissecansis a focal bone-cartilage lesion characterized by separation of anosteochondral fragment from the articular surface. Attempts to treatthis injury with allograph transplants faces the same problem of secondsurgery and disruption of the healthy tissue, as described above. Thusit would be advantageous to have available a method which would remove aneed for second surgery and yet provide a means for a cartilage and bonerepair.

The current method provides a solution to the above-outlined problems byimplanting, during the first arthroscopic surgery, a bone-inducingcomposition or a carrier comprising said composition comprising abone-inducing agents into the bone lesion and an acellular matriximplant into the cartilage lesion thereby providing, in one surgery,treatments for both the bone and cartilage defects.

D. Bone and Bone Defects

The restorative method according to this invention is additionally alsosuitable for repair of the skeletal bone lesions.

The skeletal bone lesions are lesions which are either solely or atleast partially located in the skeletal part of the bone, that is thebone placed immediately below the subchondral bone region, as seen inFIG. 1C.

FIG. 1C is a schematic representation of the deep osteochondro-skeletalbone injury extending into the skeletal bone. The figure shows thepositioning of the host cartilage, subchondral bone and the skeletalbone as well as emplacement of the acellular matrix implant into theosteochondral defect and the bone-inducing composition into thesubchondral and skeletal bone defect. The scheme shows the cartilagelesion implantation site with the acellular matrix implanted thereinsurrounded by host cartilage with underlaying bone lesion in thesubchondral bone. The bone-inducing composition or a carrier comprisingsaid composition is deposited into the bone lesion. The carrier for thispurpose may be any matrix described above but is preferably collagenous,hydrogel or a polymer of an aromatic organic acid containing structure.Emplacement of the top and bottom sealant are also shown wherein thebottom sealant separates the bone portion of the defect from thecartilage lesion such that each is treated separately using differentmeans.

In an alternative, the bone-inducing composition and/or the acellularimplant carrier comprising such composition can be used for treatment ofsimple skeletal bone defects, lesions or fractures without a need forcartilage implant.

If and when the method of the invention is used for treatment ofskeletal bone lesions, the bone-inducing composition alone orincorporated into a carrier, preferably dissolved in collagen or anotherbinding agent, is deposited directly into the skeletal bone lesion. Thebone-inducing agent is selected from the group consisting of calciumphosphate, hydroxyapatite, organoapatite, titanium oxide, demineralizedbone powder, poly-L-lactic, polyglycolic acid or a copolymer thereof anda bone morphogenic protein.

A preferred bone-inducing agent is the demineralized bone powder (DMB).DMB is derived from bone by, for example, acid extraction of the calciumphosphate. Following such extraction, the DMB retains, in addition tothe bone collagen other chemical elements found in the bone, includingthe naturally present members of TGF-β superfamily of bone developmentfactors. These factors may also be extracted by further treatment ofbone with such materials as quanidine hydrochloride. When thesenaturally occurring TGF-βs are present in the DMB, no furtherbone-inducing agents are needed to be present because DMB has a porousmicrostructure suitable for bone formation.

It is to be understood that the DMB itself is very light powder andtherefore, it is preferably formulated in an agent having a bindingcapabilities. The most preferred binding agent is collagen orcollagen-like agents, hydrogels, alginates, etc.

II. An Acellular Matrix Implant for Treatment of Cartilage Lesions

The current invention provides a method for treatment of injured,damaged, diseased or aged cartilage. To this end, the method involvesimplantation of the acellular matrix implant into the injured, damaged,diseased or aged cartilage at a site of injury or at a site of a defectcaused by disease or age, in a single surgery. The acellular matriximplant is a collagenous construct or a polymer of an aromatic organicacid comprising various components as described below.

A. Preparation of an Acellular Matrix Implant

Preparation of the acellular matrix implant for implanting into thecartilage lesion involves preparation of acellular support matrix,typically a collagenous scaffold or sponge, thermo-reversible gelationhydrogel or a polymer of an aromatic organic acid and implanting saidmatrix into the cartilage defect in situ.

The acellular matrix implant, such as the one seen in FIG. 2A, isprepared according to the method of the invention and implanted intoartificially generated lesions in a swine's knee weight bearing region.FIG. 2A is an image of an actual acellular matrix sponge implant usedfor implantation, here held in the forceps. The sponge has a size of 5mm in diameter and 1.5 mm in thickness and comprises a composition ofcollagen sponge and collagen gel having pores of sizes from about200-400 μm (FIG. 2B). When the sponge is implanted into the lesion,chondrocytes are activated and migrate into the porous structure of thesponge where they begin to secrete a new extracellular matrix ultimatelyreplacing the collagen sponge and gel with the new hyaline cartilage.The sponge and gel naturally biodegrade and are metabolically removedfrom the lesion.

FIG. 2B is a cross-side view scheme of a honeycomb structure of theacellular matrix sponge seen in FIG. 2A illustrating a relativepositioning of the collagen sponge, collagen gel and pores within theacellular matrix sponge.

The matrices of the acellular matrix implant deposited into the lesionare comprised of biodegradable materials which permit said implant tofunction for certain period of time needed for formation of the hyalinecartilage. Such biodegradable materials are subsequently biodegraded andmetabolically removed from the site of implantation leaving, if any,only non-toxic residues. These materials were additionally found topromote formation of the superficial cartilage layer which covers thelesion containing the implant thereby protecting a newly formed hyalinecartilage. The biodegradable materials may additionally include enzymes,such as metalloproteinases, paracrine or autocrine growth hormones,GAG-lyases and such like enzymes, soluble protein mediators and othersupplements. Presence or addition of these materials may enhanceactivation of mature, metabolically active but non-dividing chondrocytespresent in the surrounding native host cartilage and migration of thesechondrocytes from the native host cartilage surrounding the lesioncavity into said acellular matrix implant emplaced within said lesion.

The present invention thus concerns a discovery that when the acellularmatrix implant according to the invention is implanted into a cartilagedefect, under conditions described below, the older inactivechondrocytes residing within the surrounding native cartilage areinduced to migrate into the defect where these chondrocytes areactivated from static non-dividing stage to an active stage where theydivide, multiply, promote growth of the extracellular matrix andgenerate a new hyaline cartilage in situ. Following the implantation ofthe acellular matrix implant, the cartilage defect is quickly repaired,particularly in the young individuals, by chondrocyte migration and byformation of the extracellular matrix supported by themetalloproteinases naturally present in sufficient amounts in tissues ofthe young individuals. For the repair of lesions in older subjects, theGAG-lyases and metalloproteinases, growth factors and other componentsare added or incorporated into said matrix before implantation or theymay be conveniently used to coat said matrix to promote degradation ofthe injured cell.

A process for activation of chondrocytes was found to require certainperiod of time, typically from about 1 hour to about 3 weeks, typicallyonly about 6 hours to about 3 days. The process for complete replacementof the implant matrix with the hyaline cartilage typically takes fromone week to several months provided that the treated individual becomesnormally physically active subjecting said new cartilage to theintermittent hydrostatic pressure by, for example, walking, running orbiking.

B. Induction of Chondrocyte Migration

Induction of chondrocyte migration from the surrounding native cartilageinvolves biological actions of various agents either naturally presentwithin the cartilage, cartilage surrounding tissue, blood or plasma orthey are added either before, during or after the surgery to promoterelease, activation and migration of chondrocytes from the nativesurrounding host cartilage into the implant.

One of the steps in achieving the activation of the chondrocytes is theuse of sealants at the top and bottom of the articular cartilage lesion.This step results in creation of a cavity into which the acellularmatrix implant is deposited. A container-like porous property of theacellular collagenous matrix implant permits infusion and concentrationof soluble protein mediators, enzymes, growth or other factors, etc.,naturally present in the host's surrounding healthy cartilage.

Sealing of the top and bottom of the defect before and after insertionof the acellular matrix implant results in accumulation of autocrine andparacrine growth factors that are released by chondrocytes in theadjacent extracellular matrix, enabling these factors to induce cellmigration into the implant. Suitable growth factors include, amongothers, certain transforming growth factors, platelet-derived growthfactors, fibroblast growth factors and insulin-like growth factor-I.Additionally, these and other supplements, such as the GAG-lyases(matrix remodeling enzymes), may be used to coat the implant before itsinsertion into the lesion or the lesion itself may be coated.

The acellular matrix implant sequestered within the lesion cavity by thetop and bottom sealant, however, remains in flowable communication withthe adjacent cartilage. This arrangement creates conditions resulting indecrease of levels of inhibitors of the matrix remodeling enzymes, suchas tissue inhibitors of metalloproteinase-1 (TIMP-1),metalloproteinase-2 (TIMP-2) and metalloproteinase-3 (TIMP-3), at thedefect site. As a consequence, the matrix metalloproteinases (MMP-1,MMP-2, MMP-3) become accessible to enzymatic activation and degrade theadjacent extracellular matrix thereby releasing chondrocytes localizedtherein resulting in chondrocytes migration from the surrounding hostcartilage into the acellular matrix implant or coat the walls of thelesion itself with the sugar lyases.

The acellular matrix implant sealed within the lesion also becomes arepository of exogenous growth factors that pass through the bottomsealant layer in response to joint loading and hydrostatic pressure towhich the joint is subjected when undergoing a normal physical activitysuch as walking, running or biking. Consequently, in response to thehydrostatic pressure load, these factors become more concentrated withinthe defect site and chondrocytes released from adjacent areas of thesurrounding extracellular matrix migrate into the lesion with ensuingchondrocyte proliferation and initiation of the de novo extracellularmatrix synthesis within the lesion.

Moreover, the acellular matrix of the implant fills the defect with amaterial that has a reduced stiffness relative to normal articularcartilage and permits deformation of the adjacent native cartilagematrix edges thereby increasing level of shear stress further resultingin increased release of soluble mediators that indicate matrixremodeling and chondrocyte migration into the acellular matrix implant.

The presence of the acellular matrix implant sealed to the adjacentcartilage boundaries thus creates conditions by which matrix remodelingenzymes, namely matrix metalloproteinases, aggrecanases and cathepsins,become concentrated at the defect site and initiate enzymatic opening ofthe adjacent extracellular matrix so that chondrocytes may migrate intothe acellular matrix implant, be deposited within its matrix, begin todivide and proliferate and secrete the new extracellular matrix,ultimately leading to formation of normal healthy hyaline cartilage.

C. Types of Acellular Matrix Implant

The acellular matrix implant provides a structural support formigration, growth and two or three-dimensional propagation ofchondrocytes in situ. Generally, the acellular matrix is biologicallybiocompatible, biodegradable, hydrophilic and preferably has a neutralcharge.

Typically, the implant is a two or three-dimensional structuralcomposition, or a composition able to be converted into such structure,containing a plurality of pores dividing the space into a fluidicallyconnected interstitial network. In some embodiments the implant is asponge-like structure, honeycomb-like lattice, sol-gel, gel orthermo-reversible gelation hydrogel.

Typically, the implant is prepared from a collagenous gel or gelsolution containing Type I collagen, Type II collagen, Type IV collagen,gelatin, agarose, hyaluronin, cell-contracted collagens containingproteoglycans, polymers of organic aromatic acids, glycosaminoglycans orglycoproteins, fibronectins, laminins, bioactive peptide growth factors,cytokines, elastins, fibrins, synthetic polymeric fibers made ofpoly-acids such as polylactic, polyglycotic or polyamino acids,polycaprolactones, polypeptide gels, copolymers thereof and combinationsthereof. Preferably, the implant matrix is a gel, sol-gel, a polymer ofan aromatic organic acid or a polymeric thermo-reversible gel. Mostpreferably the implant matrix contains aqueous Type I collagen.

The acellular matrix implant may be of a type of sponge, scaffold orhoneycomb sponge, scaffold or honeycomb-like lattice or it may be a gel,sol-gel or thermo-reversible gel composition or it may be a polymer ofan aromatic organic acid.

The acellular matrix implant may be produced as two or three-dimensionalentities having an approximate size of the lesion into which they aredeposited. Their size and shape is determined by the shape and size ofthe defect.

a. Acellular Sponges or Sponge-Like Implants

In general, any polymeric material can serve as the support matrix,provided it is biocompatible with tissue and possesses the requiredgeometry. Polymers, natural or synthetic, which can be induced toundergo formation of fibers or coacervates, can be freeze-dried asaqueous dispersions to form sponges.

In addition to collagen, a wide range of polymers may be suitable forthe fabrication of sponges, including agarose, hyaluronic acid, alginicacid, dextrans, polyHEMA, and poly-vinyl alcohol alone or incombination.

Typically, such sponges must be stabilized by cross-linking, such as,for example, ionizing radiation. Practical example includes preparationof freeze-dried sponges of poly-hydroxyethyl-methacrylate (pHEMA),optionally containing additional molecules, such as gelatin,advantageously entrapped within. Incorporation of agarose, hyaluronicacid, or other bio-active polymers can be used to modulate cellularresponses. All these types of sponges can function advantageously asimplant matrices for the purposes of the present invention.

The gel or gel solution used for preparation of the sponge orsponge-like implant is typically washed with water and subsequentlyfreeze-dried or lyophilized to yield a sponge like matrix able toincorporate the migrating chondrocytes within the matrix. The acellularmatrix implant of the current invention acts like a porous sponge wheninfiltrated with the migrating chondrocytes wherein the cells aredistributed within the sponge pores, providing a mesh-like supportpermitting the chondrocytes to migrate and settle there, begin to divideand proliferate and secrete materials for generation of newextracellular matrix and eventually for generation of hyaline cartilagecontiguous with the existing healthy surrounding cartilage.

One important aspect of the sponge implant is the pore size of thesponge matrix. Sponges having different pore sizes permit faster orslower infiltration of the chondrocytes into said sponge, faster orslower growth and propagation of the cells and, ultimately, the higheror lower density of the cells in the implant. Such pore size may beadjusted by varying the pH of the gel solution, collagen concentration,lyophilization conditions, etc., during implant fabrication. Typically,the pore size of the sponge is from about 50 to about 500 μm, preferablythe pore size is between 100 and 300 μm and most preferably about 200μm.

The pore size of the acellular matrix implant will be selected dependingon the recipient. In the young recipient where the metalloproteinasesare present naturally and active, the pore size will be smaller as theactivated chondrocytes will rapidly proliferate through the pores andsecrete extracellular matrix. In older recipients, the pores will bebigger as the migrating chondrocytes will be sluggish and will need moretime to settle in the pores and proliferate.

An exemplary acellular matrix implant made of collagen is seen in FIG.2. FIG. 2A is an example image of acellular collagenous matrix implantof size 4 mm in diameter and of 1.5 mm in thickness. The seeding densityof this implant is between 300,000-375,000 chondrocytes per 25 μl volumecorresponding to about 12-15 millions cells/ml. The cell densityfollowing the implantation of the acellular matrix implant is, ofcourse, dependent on the rapidity of the migration of chondrocytes fromthe surrounding native cartilage and on their ability to divide andrapidity of their multiplication, however, the collagenous matrix of theimplant has a capacity to accommodate this range of migrating cells.

The acellular sponge may be prepared according to procedures describedin Example 1, or by any other procedure, such as, for example,procedures described in the U.S. Pat. Nos. 6,022,744; 5,206,028;5,656,492; 4,522,753 and 6,080,194 or in co-pending application Ser.Nos. 10/625,822, 10/625,245 and 10/626,459, herein incorporated byreference.

b. Acellular Scaffold or Honeycomb Implants

One type of the implant of the invention is an acellular scaffold,honeycomb scaffold, honeycomb sponge or honeycomb-like lattice. Allthese implants contain a honeycomb-like lattice matrix providing asupport structure for migrating and dividing chondrocytes. Thehoneycomb-like matrix is similar to that of the sponge described abovebut has that typical pattern of the honeycomb. Such honeycomb matrixprovides a growth platform for the migrating chondrocytes and permitsthree-dimensional propagation of the migrated and divided chondrocytesthereby providing a structural support for formation of new hyalinecartilage.

FIG. 2B is a side view scheme of honeycomb structure of acellular matrixshowing a collagen sponge and collagen gel with pore (*) size of eachcolumn of about 200-400 μm.

The honeycomb-like matrix is fabricated from a polymerous compound, suchas collagen, gelatin, Type I collagen, Type II collagen or any otherpolymer, as described above for the sponge, having a desirableproperties. In the preferred embodiment, the honeycomb-like acellularmatrix implant is prepared from a solution comprising Type I collagen.

The pores of the honeycomb-like implant are evenly distributed withinsaid honeycomb matrix to form a structure able of taking in and evenlydistributing the migrated chondrocytes.

One preferred type of acellular matrix implant is Type-I collagensupport matrix fabricated into a honeycomb-lattice, commerciallyavailable from Koken Company, Ltd., Tokyo, Japan, under the trade nameHoneycomb Sponge.

Acellular matrix implant of the invention thus may be any suitablebiodegradable structure, gel or solution, preferably containingcollagen. For the purposes of convenience in implanting, such implant istypically a gel, preferably sol-gel transitional solution which changesthe state of the solution from liquid sol to solid gel above roomtemperature. The most preferred such solution is the thermo-reversiblegelation hydrogel or a thermo-reversible polymer gel as described below.

c. Sol-Gel Acellular Matrix Implant

Another type of acellular matrix implant is the implant matrixfabricated from sol-gel materials wherein said sol-gel materials can beconverted from sol to gel and vice versa by changing temperature. Forthese materials the sol-gel transition occurs on the oppositetemperature cycle of agar and gelatin gels. Thus, in these materials thesol is converted to a solid gel at a higher temperature.

Sol-gel material is a material which is a viscous sol at temperaturesbelow 15° C. and a solid gel at temperatures around and above 37° C.Typically, these materials change their form from sol to gel bytransition at temperatures between about 15° C. and 37° C. and are in atransitional state at temperatures between 15° C. and 37°. However, bychanging the hydrogel composition, the transition temperature of thesol-gel may be predetermined to be higher or lower than those givenabove. The most preferred materials are Type I collagen containing gelsand a thermo-reversible gelation hydrogel (TRGH) which has a rapidgelation point.

In one embodiment, the sol-gel material is substantially composed ofType I collagen and, in the form of 99.9% pure pepsin-solubilized bovinedermal collagen dissolved in 0.012 N HCl, commercially available underthe trade name VITROGEN® from Cohesion Corporation, Palo Alto, Calif.One important characteristic of this sol-gel is its ability to be curedby transition into a solid gel form wherein said gel cannot be mixed orpoured or otherwise disturbed thereby forming a solid structureoptionally containing other components supporting the chondrocytesactivation and migration. Sterile collagen for tissue culture may beadditionally obtained from other sources, such as, for example,Collaborative Biomedical, Bedford, Mass., and Gattefosse, SA, St.Priest, France.

Type I collagen sol-gel is generally suitable and preferred material forfabrication of an acellular sol-gel implant.

d. Thermo-Reversible Gelation Hydrogel Implants

Additionally, the acellular matrix implant may be prepared fromthermo-reversible materials similar to sol-gel which materials, however,have much faster point of transition, without hysteresis, from sol togel and vice versa.

The thermo-reversible property is important for implantation of theacellular matrix implant into the lesion cavity as it may be implantedinto the lesion cavity in its sol state whereby filling said cavity withthe sol wherein the sol forms itself according to the exact shape of thecavity leaving no empty space or being too large or too small, as thecase may be, for a prefabricated sponge or a honeycomb lattice.Following the warming of the sol emplaced within the articular lesioncavity to the natural body temperature, the sol instantly transitionsand becomes solid gel providing a structural support for the migratingchondrocytes from the surrounding native cartilage.

One characteristic of the sol-gel is its ability to be cured ortransitioned from a liquid into a solid form and vice versa. Thisproperty may be advantageously used for solidifying the liquid orliquefying the solid gel acellular matrix implant within the cartilagelesion as well as for delivery, storing or preservation purposes of saidacellular matrix implant. Additionally, these properties of sol-gel alsopermit its use as a support matrix by changing its sol-gel transition byincreasing or decreasing temperature in the lesion, or exposing thesol-gel to various chemical or physical conditions or ultravioletradiation.

In one embodiment, the acellular matrix implant is a thermo-reversiblegelation hydrogel or gel polymer kept stored and implanted attemperatures between 5° C. and 15° C. At that temperature, the hydrogelis at a liquid sol stage and permits easy emplacement into the lesion asthe sol. Once the sol is emplaced within the lesion, the sol isnaturally or artificially subjected to higher temperature of about 30°C. and 37° C. at which temperature the liquid sol solidifies into solidgel. The gelling time is from about several minutes to several hours,typically about 1 hour. In such an instance, the solidified gel mayitself become and be used as an implant or this sol may be loaded into aseparate support matrix, such as a sponge or scaffold honeycomb implant.

The primary characteristic of the thermo-reversible gelation hydrogel(TRGH) is that upon its degradation within the body it does not leavebiologically deleterious material and that it does not absorb water atgel temperatures. TRGH has a very quick sol-gel transformation whichrequires no cure time and occurs simply as a function of temperaturewithout hysteresis. The sol-gel transition temperature can be set at anytemperature in the range from 5° C. to 70° C. by the molecular design ofthe thermo-reversible gelation polymer (TGP), a high molecular weightpolymer, of which less than 5 wt % is enough for hydrogel formation.

The thermo-reversible gelation hydrogel (TRGH), should be compressivelystrong and stable at 37° C. and below till about 32° C., that is toabout temperature of the synovial capsule of the joint which istypically below 37° C., but should easily solubilize below 30-31° C. tobe able to be conveniently changed to the sol within the lesion cavity.The compressive strength of the TRGH must be able to resist compressionby the normal activity of the joint.

The typical TRGH is generally made of blocks of high molecular weightpolymer comprising numerous hydrophobic domains cross-linked withhydrophilic polymer blocks. TRGH has a low osmotic pressure and is verystable as it is not dissolved in water when the temperature ismaintained above the sol-gel transition temperature. Hydrophilic polymerblocks in the hydrogel prevent macroscopic phase separation andseparation of water from hydrogel during gelation. These properties makeit especially suitable for safe storing and extended shelf-life.

In this regard, the thermo-reversible hydrogel is an aqueous solution ofthermo-reversible gelation polymer (TGP) which turns into hydrogel uponheating and liquefies upon cooling. TGP is a block copolymer composed oftemperature responsive polymer (TRP) block, such aspoly(N-isopropylacrylamide) or polypropylene oxide and of hydrophilicpolymer blocks such as polyethylene oxide.

Thermally reversible hydrogels consisting of co-polymers of polyethyleneoxide and polypropylene oxide are available, for example, from BASFWyandotte Chemical Corporation under the trade name of Pluronics.

In general, thermo-reversibility is due to the presence of hydrophobicand hydrophilic groups on the same polymer chain, such as in the case ofcollagen and copolymers of polyethylene oxide and polypropylene oxide.When the polymer solution is warmed, hydrophobic interactions causechain association and gelation; when the polymer solution is cooled, thehydrophobic interaction disappears and the polymer chains aredis-associated, leading to dissolution of the gel. Any suitablybiocompatible polymer, natural or synthetic, with such characteristicswill exhibit the same reversible gelling behavior.

e) Acellular Gel Implants

The acellular matrix implants of the invention may alternatively beprepared from various gel materials, such as suspending gels, notnecessarily thermo-reversible, which are commercially available and maybe suitable for use as acellular matrix implants as long as they arebiodegradable.

One example of such gel is polyethylene glycol (PEG) and itsderivatives, in which one PEG chain contains vinyl sulfone or acrylateend groups and the other PEG chain contains free thiol groups whichcovalently bond to form thio-ether linkages. If one or both partner PEGmolecules are branched (three- or four-armed), the coupling results in agel network. If the molecular weight of the PEG chains used for theimplant preparation is between 500 and 10,000 Daltons along any linearchain segment, the network will be open and suitable for receivingmigrating chondrocytes, swellable by interstitial water, and compatiblewith living chondrocytes.

The coupling reaction of PEG can be accomplished, for example, bypreparing 5 to 20% (w/v) solutions of each PEG separately in aqueousbuffers or cell culture media. Just prior to implantation, thiol, PEGand the acrylate or vinyl sulfone PEG are mixed and infused into thelesion. Gelation will begin spontaneously in 1 to 5 minutes. The rate ofgelation can be modulated somewhat by the concentration of PEG reagentand by pH. The rate of coupling is faster at pH 7.8 than at pH 6.9.Thus, by modifying the pH of the PEG containing mixture, the gelationprocess may be controlled to be faster or slower, as desired by thesurgeon. Such gels are, however, typically not degradable within thebody unless the additional ester or labile linkages are incorporatedinto the chain. PEG reagents may be purchased from Shearwater Polymers,Huntsville, Ala., USA; or from SunBio, Korea.

In a second alternative, the gelling material may be alginate. Alginatesolutions are gellable in the presence of calcium ions. This reactionhas been employed for many years to suspend cells in gels ormicro-capsules. A solution of alginate (1-2%; w/v) in culture mediadevoid of calcium or other divalent ions is mixed in a solutioncontaining calcium chloride which will gel the alginate. Analogousreactions can be accomplished with other polymers which bear negativelycharged carboxyl groups, such as hyaluronic acid. Viscous solutions ofhyaluronic acid can be gelled by diffusion of ferric ions.

f. A Polymer of an Aromatic Organic Acid Matrix

The acellular implant may also conveniently be made of a polymer of anaromatic organic acid. Polymers of this type have typically a negativecharge and are thus preferred for use as a bone-inducing compositioncarriers. However, these type of compounds may also be used and aresuitable for use as cartilage acellular implants.

G. Biodegradable Implant

The acellular matrix implant of the invention is a temporary structureintended to provide a temporary supporting for the migrating, dividing,proliferating and extracellular matrix secreting chondrocytes releasedfrom the surrounding cartilage.

Consequently, the implant of the invention must be fully biodegradable.Whether it is a sponge, honeycomb lattice, sol-gel or TRGH, in time, thedelivered implant is disintegrated or incorporated into the existingcartilage and the TRGH is subsequently degraded leaving no undesirabledebris behind.

Overall, any of the acellular matrix implants for cartilage defectsdescribed above is suitable for implantation into a cartilage lesion ofany size and shape and provides a support for a structural rebuilding ofthe cartilage by migrating chondrocytes therein from the surroundinghealthy host cartilage. The implantation of the implant of the inventionresults in the generation of normal healthy hyaline cartilage and incomplete healing of the cartilage defect.

III. Osteochondral Defects and Treatment Thereof

Lesions of the articular cartilage are often accompanied by lesions ofthe underlying bone. Such defects are thus a composite of cartilage andunderlying bone. These defects are herein cumulatively calledosteochondral defects.

A. Method for Treatment of Osteochondral Defects

The osteochondral defects are caused by injury of the cartilage andbone. The cartilage and bone are histologically two different connectivetissues, as described above. Consequently, it is not possible toeffectively treat both using the same methods and means and suchtreatment is thus complex and more difficult than a treatment of thecartilage lesion or chipped bone alone. Furthermore, bone developmentcan influence cartilage development such that it acts as a barrier tofurther cartilage development during its critical developmental stages.

In one attempt to treat these complex injuries, a mosaicplasty techniquewas developed. The mosaicplasty, as already mentioned above, involves aremoval of grafts from the healthy tissue and plugging such grafts intothe both bone and cartilage lesions. An obvious defect of this techniqueis that in order to treat the injured site, surgeon has to remove,during the open surgical procedure, a healthy tissue from another sitethereby disrupting the healthy tissue in the process.

When, however, the method of the current invention is used to treatthese complex osteochondral injuries, it is possible to treat both thebone and cartilage lesions during the same surgery without need toremove and disturb the healthy tissue and/or undergo multiple surgeriesrequired, for example, for allograft transplantation and othertechniques.

The current method permits such dual treatment simultaneously byimplantation of, in combination, an acellular matrix implant and abone-inducing composition or a carrier comprising said compositioncomprising a bone-inducing agents further, preferably, in combinationwith biologically acceptable sealants.

In practice, during the same surgery, the surgeon first debrides bothlesions and deposits the bone-inducing composition or a carriercomprising said composition into the bone lesion and covers said bonelesions with one or several layers of a biologically acceptable sealant,preferably a modified highly polymerizable sealant selected from thosedescribed below in section IV. After the sealant polymerizes, typicallywithin several minutes, preferably between 0.5 and 5 minutes, theacellular matrix implant is deposited into the cartilage lesion andcovered with yet another layer of the sealant, herein called the topsealant. In this way, the bone-inducing composition or a carriercomprising said composition is sequestered within the bone lesion andthe bone forming agents, such as, for example, demineralized bonepowder, calcium phosphates, calcium citrate, hydroxyapatite,organoapatite, titanium oxide, polyacrylate, alone or in combination,and a bone morphogenic protein and/or other known bone-inducing agentsact as inducement for osteoblast migration from the surrounding bonewithout interference from the acellular matrix implant. As a consequenceof this separation of the bone and cartilage lesion, there is noinvasion of the hyaline cartilage or formation of fibrocartilage in thebone lesion.

Conversely, when the acellular implant is separated from thebone-inducing composition or a carrier comprising said composition,there is no interference from any of the bone-inducing agents with thechondrocyte migration, extracelular matrix formation and generation ofthe hyaline cartilage. Each the bone and the cartilage are treatedseparately and yet simultaneously during one arthroscopic surgery.

The sealant may be deposited, preferably, as is, that is without anyadditional agents being added, or it may be added to the bone-inducingcomposition or a carrier comprising said composition, if desirable.

The bone-inducing composition or a carrier comprising said compositiondeposited within the bone defect covered with the sealant is left in thelesion in order to achieve the bone reconstruction and growth. Both thecomposition and the sealant are aiding in a bone natural healing.

The acellular matrix implant implanted within the cartilage defectseparated from the bone lesion by a layer of the sealant and coveredwith the top sealant is left in the cartilage lesion until itbiodegrades when the hyaline cartilage replacement is formed in order toachieve the chondrocyte migration and formation of extracellular matrix.

A typical process for repair of osteochondral defects is the cleaningand debridement the osteochondral defect, depositing the bone-inducingcomposition or a carrier comprising said composition containing thebone-inducing agents, up to the upper limit of the lesion in subchondralbone, applying a layer of the sealant over the composition and lettingthe sealant to polymerize. After the sealant polymerizes, typically infrom about 0.2 to about 10 minutes, preferably about 0.5-5 minutes, thesurgery proceeds with implanting the acellular matrix implant into thecartilage lesion, as described above. The cartilage lesion containingthe implant is then covered with yet another layer of the sealant (topsealant) to seal and protect the wound from the exterior.

The above described procedure is particularly suitable for treatment ofosteochondral injuries as it permits dual treatment under differentconditions being implemented during the same surgery.

One specific case of osteochondral defects is osteochodritis dissecans,where a focal lesion of the bone and cartilage results in a loose ortotally dislocated osteochondral fragment. Currently the only availabletreatment requires three independent surgeries including biopsyharvesting of periosteum (first surgery), culturing cells, removal ofthe loose fragment (second surgery), introduction of the cultured cellsinto the lesion and bone-grafting (third surgery).

The current method, as described above, or modified to include a step ofthe fragment removal, during a single surgery, eliminates a need for twoor three surgeries, as all steps necessary for repair of theosteochondritis dissecans are performed at the same time during onesurgery.

B. Bone-Inducing Agents

Bone-inducing agents are compounds or proteins having a definite abilityto promote formation of the bone.

The most suitable bone forming agents are demineralized bone powder(DMP), calcium phosphate, calcium citrate, hydroxyapatite,organoapatite, titanium oxide and growth factors, namely a group ofgrowth factors known as bone morphogenic proteins, fibroblast growthfactor (FGF), platelet derived growth factor (PDFG), epithelial growthfactor (EGF), glioma derived factor (GDF) and transforming growth factorbeta-1 (TGF-β1). These growth factors may be used individually and/or incombination with each other or with other bone-inducing factors.

The demineralized bone powder is particularly suitable to be used as abone-inducing composition or as a bone-inducing carrier and no othercompounds are needed to serve as bone inducer or supporting structureand necessary because the demineralized bone powder mimics microporousstructure of the bone. Before depositing the DMB into the bone orsubchondral bone lesion, the DMB may be conveniently dissolved incollagen or some other adhesive fluid or hydrogel which will permit itsdeposition into the lesion but itself will have no bone-inducingfunction. The used amount of DMB is such that is makes a concentratedhighly viscous paste. The used amount depends on the structure and grindof the DMB.

Bone morphogenic proteins are typically identified by the abbreviationBMP and are further distinguished from each other by numbering, such asBMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-14. Some of themare further identified by a generic name, such as, for example BMP-3 iscalled osteogenin, BMP-3B is GDF-10, etc. The bone morphogenic proteinsare administered generally in concentration (per carrier volume orweight) of from about 0.01 to about 5 mg/cm³, preferably from about 0.1to about 1.5 mg/cm³ or from about 0.01 mg/g to about 5 mg/g, preferablyfrom about 0.1 mg to about 2.5 mg/g.

C. Bone-Inducing Composition

The bone-inducing composition or a carrier comprising said compositionof the invention comprises one or several bone-inducing agents as listedabove, in concentrations as disclosed. The bone-inducing composition maybe administered as a powder, solution, gel, sol-gel, TRGH mixed inconcentration given above or incorporated into a structure similar tothat of the acellular implant, pre-prepared and implanted into the bonelesion or fracture. The composition prepared as TRGH, for example, isprepared as a sol solution and administered as such. The solsubsequently changes its state into the gel filling out the whole bonelesion. The bone-inducing agents may also be dissolved in PEG, collagen,alginate, etc., and deposited as such. It could also be soaked up in asecond sponge system like the acellular matrix sponge described above.

The preferred mode for the deposition of the bone-inducing agents intothe osteochondral or bone lesion is to dissolve the agent in a gel, suchas diluted collagen, alginate and such like gels.

D. Bone-Inducing Carrier

A bone-inducing carrier or a carrier comprising bone-inducingcomposition is a carrier compound which is suitable for depositing saidbone-inducing composition comprising at least one bone-inducing agentor, preferably, a combination of several agents into a bone lesion.Typically, the carrier will be a biodegradable porous matrix, hydrogel,sponge, honeycomb, or scaffold having large pores from about 50 to about150 μm, which pores encourage migration of osteoblast. The carrier willalso have an interconnecting small pores of about 0.1 to about 10 μmwhich connect the large pores, permit the osteoblast to settle withinthe carrier and provide a supporting matrix and connectingmicrostructure for supply of nutrient and other factors therebypermitting the bone formation. The surface of such carrier might benegatively charged encouraging pseudopod attachment of osteoblasts andtheir migration into the carrier resulting in the bone formation.

IV. Biologically Acceptable Sealants

Generally, the implant is implanted into the cartilage or bone lesionand between at least two layers, of top and bottom of biologicallyacceptable adhesive sealants.

In practice, the first (bottom) layer of the sealant is introduced intothe lesion and deposited at the bottom of the lesion. The firstsealant's function is to prevent entry and to block the migration ofsubchondral and synovial cells of the extraneous components, such asblood-borne agents, cell and cell debris, etc. Before the implant isdeposited, such debris could interfere with the integration of theacellular matrix implant. The second function of the first sealant is tocontain enzymes, hormones and other components which are naturallypresent in the lesion and which are needed for chondrocyte activation,migration, secretion of other agents and proliferation of newly formedextracellular matrix and hyaline cartilage. Then the acellular matriximplant is implanted over the first sealant. The second (top) sealantlayer is placed over the acellular matrix implant. The presence of boththese sealants in combination with the acellular matrix implant resultsin successful activation of chondrocytes, their migration andintegration into the implant matrix and ultimately in new formation ofjoint hyaline cartilage.

A. A First—Bottom Sealant

In a method for treatment of cartilage lesions, the first (bottom)sealant forms an interface between the introduced implant and the nativetissue, such as subchondral bone or cartilage. The first sealant,deposited at the bottom of the lesion, must be able to contain migratingchondrocytes within the lesion, to protect the implant from influx ofundesirable agents and to prevent chondrocyte migration into thesub-chondral space. Additionally, the first sealant prevents theinfiltration of blood vessels and undesirable cells and cell debris intothe implant and it also prevents formation of the fibrocartilage.

In a method for treatment of osteochondral defects, the first (bottom)sealant forms a barrier between the cartilage lesion and the bonelesion. Because these two defects are in two qualitatively differenttissues they require different treatments. As described above, the bonelesion is treated with the bone-inducing composition or a carriercomprising said composition while the cartilage lesion is treated withthe acellular matrix implant. Moreover, it is not desirable that theenzymes present in the cartilage lesion activating chondrocyte migrationmix with the bone-inducing agents and growth factors needed for bonelesion repair. When there is no separation of one tissue from another,it can easily end up with, for example, the fibrocartilage ingrowinginto the bone area and, in such an instance, instead of bone beingreplaced with the bone, it is replaced with the inferior fibrocartilage.Consequently, for treatment of osteochondral defects, the bottom sealantis deposited over the bone lesion filled with the bone-inducingcomposition or a carrier comprising said composition separating the bonelesion from the cartilage lesion implanted with the acellular implant.In this way, each the acellular implant and the bone-inducingcomposition can work independently and without interference from theother.

B. A Second—Top Sealant

The second (top) sealant acts as a protector of the acellular matriximplant or the lesion cavity on the surface and is typically depositedover the lesion after the implant is deposited therein and in this wayprotects the integrity of the lesion cavity from any undesirable effectsof the outside environment, such as invading cells or degradative agentsand seals the acellular matrix implant gel in place after its depositiontherein.

The second sealant also acts as a protector of the acellular implantimplanted within a cavity formed between the two sealants. In this way,the second sealant is deposited after the implant is deposited over thefirst sealant and seals the implant within the cavity or it may bedeposited over the space holding gel before the implant deposition.

The third function of the second sealant is as an initiator or substratefor the formation of a superficial cartilage layer.

Performed studies described below confirmed that when the second sealantwas deposited over the cartilage lesion, a growth of the superficialcartilage layer occurred as an extension of the native superficialcartilage layer. This superficial cartilage layer was particularlywell-developed when the lesion cavity was filled with thethermo-reversible gel or sol gel thereby leading to the conclusion thatsuch gel might provide a substrate for the formation of such superficialcartilage layer.

C. Top and Bottom Sealant Properties

The first bottom or second top sealant used according to an embodimentof the invention must possess the following characteristics:

Sealant must be biologically acceptable, easy to use and possessrequired adhesive and cohesive properties.

The sealant must be biologically compatible with tissue, be non-toxic,not swell excessively, not be extremely rigid or hard, as this couldcause abrasion of or extrusion of the sealant from the tissue site, mustnot interfere with the formation of new cartilage, or promote theformation of other interfering or undesired tissue, such as bone orblood vessels and must be bioresorbable and biodegradable by anyacceptable metabolic pathway, or be incorporated into the newly formedhyaline cartilage tissue.

The sealant must rapidly gel from a flowable liquid or paste to aload-bearing gel within 3 to 15 minutes, preferably within 3-5 minutes.However, the sealant must not gel or polymerize too rapidly as it wouldcause problems with its even distribution over the lesion. Gellingfaster than 30 seconds is undesirable. Longer gelation times are notcompatible with surgical time constraints. Additionally, the overallmode of use should be relatively simple because complex and lengthyprocedures will not be accepted by surgeons.

Adhesive bonding is required to attach the sealant formulation to tissueand to seal and support such tissue. Minimal possessing peel strengthsof the sealant should be at least 3 N/m and preferably 10 to 30 N/m.Additionally, the sealant must itself be sufficiently strong so that itdoes not break or tear internally, i.e., it must possess sufficientcohesive strength, measured as tensile strength in the range of 0.2 MPa,but preferably 0.8 to 1.0 MPa. Alternatively, a lap shear measurementwhich define the bond strength of the formulation should have values ofat least 0.5 N/cm² and preferably 1 to 6 N/cm².

Sealants possessing the required characteristics are typicallypolymeric. In the un-cured, or liquid state, such sealant materialsconsist of freely flowable polymer chains which are not cross-linkedtogether, but are neat liquids or are dissolved in physiologicallycompatible aqueous buffers. The polymeric chains also possess sidechains or available groups which can, upon the appropriate triggeringstep, react with each other to couple or cross-link the polymer chainstogether. If the polymer chains are branched, i.e., comprising three ormore arms on at least one partner, the coupling reaction leads to theformation of a network which is infinite in molecular weight, such asfor example, a gel.

The formed gel has cohesive strength dependent on the number ofinter-chain linkages, the length expressed as molecular weight of thechains between links, the degree of inclusion of solvent in the gel, thepresence of reinforcing agents, and other factors. Typically, networksin which the molecular weight of chain segments between junction points(cross-link bonds) is between 100-500 Daltons are tough, strong, and donot swell appreciably. Networks in which the chain segments are between500-2500 Daltons swell dramatically in aqueous solvents and becomemechanically weak. In some cases the latter gels can be strengthened byspecific reinforcer molecules; for example, the methylated collagenreinforces the gels formed from 4-armed PEGs of 10,000 Daltons (2500Daltons per chain segment).

The gel's adhesive strength permits bonding to adjacent biologicaltissue by one or more mechanisms, including electrostatic, hydrophobic,or covalent bonding. Adhesion can also occur through mechanicalinter-lock, in which the uncured liquid flows into tissue irregularitiesand fissures, then, upon solidification, the gel is mechanicallyattached to the tissue surface.

At the time of use, some type of triggering action is required. Forexample, it can be the mixing of two reactive partners, it can be theaddition of a reagent to raise the pH, or it can be the application ofheat or light energy.

Once the sealant is in place, it must be non-toxic to adjacent tissue,and it must be incorporated into the tissue and retained permanently,degraded in situ, or be naturally removed, usually by hydrolytic orenzymatic degradation. Degradation can occur internally in the polymerchains, or by degradation of chain linkages, followed by diffusion andremoval of polymer fragments dissolved in physiological fluids.

Another characteristic of the sealant is the degree of swelling itundergoes in the tissue environment. Excessive swelling is undesirable,both because it creates pressure and stress locally, and because aswollen sealant gel losses tensile strength, due to the plasticizingeffect of the imbibed solvent which, in this case, is physiologicalfluid. Gel swelling is modulated by the hydrophobicity of the polymerchains. In some cases it may be desirable to derivatize the base polymerof the sealant so that it is less hydrophilic. For example, one functionof methylated collagen containing sealant is presumably to controlswelling of the gel. In another example, the sealant made frompenta-erythritol tetra-thiol and polyethylene glycol diacrylate can bemodified to include polypropylene glycol diacrylate, which is lesshydrophilic than polyethylene glycol. In a third example, sealantscontaining gelatin and starch can also be methylated both on the gelatinand on the starch, again to decrease hydrophilicity.

D. Suitable Sealants

Sealants suitable for purposes of this invention include the sealantsprepared from gelatin and dialdehyde starch triggered by mixing aqueoussolutions of gelatin and dialdehyde starch which spontaneously react andgel.

In general, a sealant useful for the purposes of this application hasadhesive, or peel strengths at least 10 N/m and preferably 100 N/cm; itneeds to have tensile strength in the range of 0.2 MPa to 3 MPa, butpreferably 0.8 to 1.0 MPa. In so-called “lap shear” bonding tests,values of 0.5 up to 4-6 N/cm² are characteristic of strong biologicaladhesives.

Such properties can be achieved by a variety of materials, both naturaland synthetic. Examples of suitable sealant include gelatin anddi-aldehyde starch described in PCT WO 97/29715, 4-armed pentaerythritoltetra-thiol and polyethylene glycol diacrylate described in PCT WO00/44808, photo-polymerizable polyethylene glycol-co-poly(a-hydroxyacid) diacrylate macromers described in U.S. Pat. No. 5,410,016,periodate-oxidized gelatin described in U.S. Pat. No. 5,618,551, serumalbumin and di-functional polyethylene glycol derivatized withmaleimidyl, succinimidyl, phthalimidyl and related active groupsdescribed in PCT WO 96/03159.

Another acceptable sealant is made from a copolymer of polyethyleneglycol and polylactide, polyglycolide, polyhydroxybutyrates or polymersof aromatic organic amino acids and sometimes further containingacrylate side chains, gelled by light, in the presence of someactivating molecules.

The acceptable sealant made from periodate-oxidized gelatin remainsliquid at acid pH, because free aldehyde and amino groups on the gelatincannot react. To trigger gelation, the oxidized gelatin is mixed with abuffer that raises the pH to pH at which the solution gels.

Still another sealant made from a 4-armed pentaerythritol thiol and apolyethylene glycol diacrylate is formed when these two neat liquids(not dissolvable in aqueous buffers) are mixed.

Another type of the suitable sealant is 4-armed polyethylene glycolderivatized with succinimidyl ester and thiol plus methylated collagenin two-part polymer compositions that rapidly form a matrix where atleast one of the compounds is polymeric, such as polyamino acid,polysaccharide, polyalkylene oxide or polyethylene glycol and two partsare linked through a covalent bond, for example a cross-linked PEG withmethyl collagen, such as a cross-linked polyethylene glycol hydrogelwith methyl-collagen, as described in U.S. Pat. Nos. 6,312,725B1 and6,624,245B2, hereby incorporated by reference. One drawback of the typeof the bioadhesive described therein is that it gels and/or bondsextremely fast upon contact with tissue, particularly with tissuecontaining collagen. Consequently, this type of bioadhesive, which isdesigned for rapid gelling or bonding during vessel or tissue injurytypically needs to be modified in order to prolong the gelling and/orbonding time to be suitable for use as a sealant of the invention.

One group of suitable sealants comprises albumin. Albumin containingsealants typically comprise at least human or bovine serum albuminconjugated with a cross-linking agent. The cross-linking agent may beselected from the group consisting of glutaraldehyde, amino acids,polypeptides and proteins. Further modification may include conjugationwith a fibrous protein, such as collagen or with a gel compound althoughthis portion of the sealant is, in the current invention, generallyprovided by the support matrix of the invention. Sealants andbioadhesives or portions thereof which fall within a category of thistype of suitable sealants are disclosed in U.S. Pat. Nos. 6,310,036;6,217,894 and 6,685,726, hereby incorporated by reference.

It is worth noting that it is not the presence or absence of particularprotein or polymer chains, such as gelatin or polyethylene glycol, whichnecessarily governs the mechanical strength and degradation pattern ofthe sealant. The mechanical strength and degradation pattern arecontrolled by the cross-link density of the final cured gel, by thetypes of degradable linkages which are present, and by the types ofmodifications and the presence of reinforcing molecules, which mayaffect swelling or internal gel bonding.

The first and second sealant, or the sealant used for separation of thebone and cartilage lesions, must be a biologically acceptable, typicallyrapidly gelling and polymerizable synthetic compound having adhesive,bonding and/or gluing properties, and is typically a hydrogel, such asderivatized polyethylene glycol (PEG) which is preferably cross-linkedwith a collagen compound, typically alkylated collagen. The sealant usedfor separation of the bone and cartilage lesions should polymerizerapidly in order to permit surgeon to continue with the surgery withoutany delay. For the purposes of this invention, the sealant should have atensile strength of at least 0.3 MPa.

Additionally, the sealant may be two or more polymer compositions thatrapidly form a matrix where at least one of the compounds is polymer,such as, polyamino acid, polysaccharide, polyalkylene oxide orpolyethylene glycol and two parts are linked through a covalent bond andcross-linked PEG with collagen. The sealant of the invention typicallygels and polymerizes within about 0.5 to about 5 minutes upon contactwith tissue, particularly with tissue containing collagen.

The second sealant or the sealant used for separation of the bone andcartilage lesions may or may not be the same as the first sealant andthe first and second sealants may be utilized as a barrier between thebone and cartilage lesions but the different sealant may also be usedfor this purpose.

For the use in the current invention, the sealant is slowly polymerizedin situ after its deposition at the bottom of the lesion or between thebone-inducing composition and acellular implant. Such slowpolymerization is necessary to avoid uneven distribution of the sealantover the bottom of the lesion and also to avoid the random and unevenaccumulation of the sealant on some parts of the surface while leavingother parts of the bottom surface uncovered. The primary function of thesealant is to protect the acellular implant from undesirable effects ofmigrating cells, tissue debris and various factors present in the bloodor serum, as already discussed previously. Consequently, its evendistribution over the bottom of the lesion or over the bone-inducingcomposition is of great importance and to achieve such evendistribution, the polymerization of the sealant must not be too slow ortoo rapid in order to reach the bottom of the lesion, cover it and thenpolymerize in situ and still meet surgeon's time constraints. Forarthroscopic surgery and implantation of the acellular implant, thesealant polymerization at the bottom of the implant site needs to occurbetween 30 seconds and about 5 minutes, preferably between 30 secondsand about 3 minutes.

V. Method for Formation of Superficial Cartilage Layer Over theAcellular Matrix Implant

An accompanying aspect of this invention is a finding that when theacellular matrix implant produced according to procedures describedabove is implanted into a cartilage lesion cavity and covered with abiocompatible adhesive top sealant, the resulting combination leads to aformation of a superficial cartilage layer completely overgrowing saidlesion.

In practice, the method for formation of the superficial cartilage layercomprises several steps. First, the bottom of the lesion is covered witha first, bottom sealant deposited as polymerizable solution. Followingthe sealant polymerization, the acellular matrix implant is implantedinto said lesion and a second, top sealant is deposited over theimplant. In one embodiment, the implant may be a thermo-reversible gelwhich easily changes from sol to gel at the body temperature therebypermitting an external preparation and delivery of the implant into thelesion. The gel is then covered with the top sealant which promotesformation of the superficial cartilage layer overgrowing the cartilagelesion thereby sequestering the implant within the lesion and protectingit from outside environment.

The superficial cartilage layer begins to form very quickly after theimplant is implanted into the cartilage lesion and covered with the topsealant layer. As shown in FIG. 6, two weeks after acellular matriximplantation superficial cartilage layer was observed on the surface ofacellular matrix implanted site. FIG. 6 shows arthroscopic evaluationtwo weeks after the defect was made in the femoral condyle where thesuperficial cartilage layer is clearly visible compared to untreatedempty defect made at the same time, seen in FIG. 5.

The top sealant gives support and promotes formation of the superficialcartilage layer in some instances further assisted by the gel componentsof the matrix. At the time when the implant matrix is completelydegraded and the new hyaline cartilage is formed in the defect, thesuperficial cartilage layer completely covers and insulates the newlyformed cartilage similarly to a synovial membrane naturally present andcovering the joints. The second top sealant is eventually alsobiodegraded and removed from the site, not however, until thesuperficial cartilage layer, a synovial-like membrane, has formed over.

VI. Method for Use of Acellular Matrix Implant

The method for repair and restoration of damaged, injured, diseased oraged cartilage to a functional cartilage is based on implantation of anacellular matrix implant into a cartilage lesion.

The method for use of the acellular matrix implant in these treatmentscomprises following steps:

a) Preparing an Acellular Matrix Implant

The first step involves preparation of the acellular matrix implant forimplanting into the cartilage lesion. Preparation of acellular matriximplants is described in greater detail in sections II.A.

b) Selecting and Depositing the First and Second Sealant into theCartilage Lesion

The second step is optional and involves selection and depositing bottomand/or top sealant layers into a cartilage lesion.

Specifically, this step involves deposition of the first sealant at thebottom of the cartilage lesion and the second sealant over the acellularmatrix implant. The first and the second sealants can be the same ordifferent, however, both the first and the second sealants must havecertain definite properties to fulfill their functions.

The bottom sealant, deposited into the lesion before the acellularmatrix implant is introduced, acts as a protector of the lesion cavityintegrity. It protects the lesion cavity from contamination byextraneous substances such as blood and tissue debris. It protectsintegrity of the naturally present enzymes and other mediators neededfor and involved in formation of extracellular matrix and activation ofchondrocytes and their migration from a surrounding host cartilage intothe acellular implant implanted in the lesion. It also protects thelesion cavity from formation of the fibrocartilage.

The top sealant deposited over the implant and effectively sealing thelesion from external environment acts as a protector of the lesioncavity as well as a protector of the implant deposited within a lesioncavity formed between the two sealants as well as an initiator of theformation of the superficial cartilage layer.

c) Implanting the Acellular Matrix Implant

Next step in the method of the invention comprises implanting saidacellular matrix implant into a lesion cavity formed between two layersof sealants.

The implant is preferably deposited into said lesion cavity after thebottom sealant is deposited but before the top sealant is deposited overit or the implant may be deposited into the lesion cavity without thebottom sealant being deposited there and then covered with the topsealant.

d) Generation of the Superficial Cartilage Layer

A deposition of the top sealant over the acellular matrix implant leadsto sealing of the lesion cavity and overgrowth of said cavity with asuperficial cartilage layer.

Typically, a biologically acceptable top sealant is deposited over theacellular matrix implant implanted into the lesion cavity. The secondsealant acts as an initiator for formation of the superficial cartilagelayer which in time completely overgrows the lesion and stronglyresembles a healthy synovial membrane. In several weeks or months,usually in about two weeks, the superficial cartilage layer completelycovers the lesion, protects the implant, migrating, dividing andproliferating chondrocytes and newly secreted extracellular matrix.Protecting the implant from extraneous environment permits integrationof the newly formed cartilage tissue into the native surroundingcartilage substantially without formation of fibrocartilage.

Formation of the superficial cartilage layer is thus a very importantaspect of the healing of the cartilage and its repair and regeneration.

VII. Method for Treatment of Cartilage Lesions

The method for treatment of damaged, injured, diseased or aged cartilageaccording to the invention is suitable for healing of cartilage lesionsdue to acute injury by providing conditions for regeneration of thehealthy hyaline cartilage and for its integration into the surroundingnative cartilage.

The method generally encompasses several novel features, namely,fabrication of a biologically acceptable biodegradable acellular matriximplant, selecting and depositing a top and bottom adhesive sealants tothe lesion and the implantation of the acellular matrix implant within acavity generated by two sealants, a formation of the superficialcartilage layer covering the lesion and protecting the integrity of theacellular matrix implant deposited therein, and providing conditions foractivation, migration, dividing and proliferation of chondrocytes andfor secretion of extracellular matrix ultimately leading to formation ofthe new hyaline cartilage and its integration into the native cartilage.

The method generally comprises steps:

a) fabrication of the acellular matrix implant according to the abovedescribed procedures;

b) debridement an articular cartilage lesion in surgical procedure;

c) during the debridement, preparing the lesion for implantation of theacellular matrix implant by depositing the bottom sealant at the bottomof the lesion thereby insulating said cavity from the surroundingtissue;

d) implanting the acellular matrix implant into said cavity formed bythe polymerized bottom sealant to allow the activated and migratingchondrocytes to proliferate within said implant;

e) depositing the top sealant over the lesion, and thereby sealing saidimplant within the cavity formed between the two sealant layers;

f) optionally introducing enzymes, hormones, growth factors, proteins,peptides and other mediators into said sealed cavity by incorporatingthem into the acellular matrix, or coating said matrix with them,introducing them separately or generating conditions for their transportor transfer through the bottom sealant; and

g) following the surgery, subjecting an individual undergoing a surgeryfor repair of said lesion to a normal physical activity therebynaturally providing an intermittent hydrostatic pressure which was shownto promote formation of the healthy hyaline cartilage and itsintegration into the surrounding native intact cartilage.

There are several advantages of the current method.

The main advantage of this method is that the acellular matrix implantis prepared beforehand and is implanted during the first and onlysurgery where the cleaning and debridement is immediately followed byimplantation of the acellular matrix implant.

Second, the acellular implant avoids immunological reactions to developas there is/are no foreign tissue or cells involved because the implantis wholly synthetic and acellular.

The method using the acellular matrix implant permits athree-dimensional expansion of chondrocytes and extracellular matrix.

The deposition of the top sealant layer resulting in formation ofsuperficial cartilage layer constitutes a substitute for synovialmembrane and provides the outer surface of healthy articular cartilageovergrowing, protecting, containing and providing critical metabolicfactors aiding in protecting the implant and activated migratingchondrocytes in the lesion. The superficial cartilage layer also preventinvasion of pannus as seen in FIGS. 10A, 10B, 11A and 11B compared withFIGS. 8A, 8B, 9A and 9B, where the presence of the invading pannus isclearly visible. In some instances, a selection of the thermo-reversiblegel may be crucial as certain TRGH may function as a promoter for growthof the superficial cartilage layer without a need to apply the topsealant.

Deposition of the bottom sealant layer protects the integrity of thelesion after cleaning during surgery and prevents migration ofsubchondral and synovial cells and cell products thereby creating milieufor formation of healthy hyaline cartilage from the activated migratingchondrocytes into the acellular matrix implant and also preventingformation of the fibrocartilage.

The method further permits said acellular matrix implant to be enhancedwith hyaluronic acid or other components or mediators named above,typically added in about 5 to about 50%, preferably about 20% (v/v),wherein such hyaluronic acid or such other components act as enhancersof the matrix-forming characteristics of the gel and also as a hydrationfactor in the synovial space in general and within the lesion cavity inparticular.

Further, the method is very versatile and any of the implant typevariations may be advantageously utilized for treatment of a specificcartilage, osteochondral or bone injury, damage, aging or disease.

For treatment of the cartilage, a subject is treated, according to thisinvention, with a prepared acellular matrix implant implanted into thelesion, the implant is left in the lesion covered with the top sealantfor as long as needed. Usually, during the two-three months followingthe surgery and implant implantation the new hyaline cartilage is formedand integrated into the native surrounding host cartilage. Typicallyalso, there is no need for any further surgical or other intervention,as during these two-three months, at a normal physical activity, such aswalking, running or biking, etc., a sufficient hydrostatic pressure isapplied to the lesion to initiate and promote formation of the hyalinecartilage fully integrated into the native cartilage. Such cartilagewill then become a fully functional cartilage covered with a superficialcartilage layer which eventually grows into or provides the same type ofsurface as a synovial membrane of the intact joint.

Finally, the method also permits replacement of the age worn-out ordiseased osteoarthritic cartilage by the regenerated hyaline-likecartilage when treated according to this invention.

The implantation protocol may assume any variation described above orpossible within the realm of this invention. It is thus intended thatevery and all variations in the treatment protocol, the types of theimplants, use of one or two sealants, implantation process, selection ofadded mediators and not the least the normal physical activity of theindividual are within the scope of the current invention.

VIII. Method for Treatment of Bone or Osteochondral Defects

The method for treatment of osteochondral defects is typically practicedin conjunction with treatment of cartilage. The method for treatment ofbone defects and lesions may be practiced in conjunction withosteochondral defects or separately without steps involving depositionof the acellular implant into the cartilage.

A. Osteochondral Defects

Due to its anatomical arrangement where the subchondral bone islocalized directly beneath the injured cartilage and the injury is boththe injury to the cartilage and to the subchondral bone or subchondralskeletal bone, the method for treatment of osteochondral defects is anextension of the method for treatment of cartilage lesions described insection VII, with exception that during the step c) of that method, thesurgeon, after debridement, deposits into the subchondral bone lesion abone-inducing composition or a carrier comprising said compositiontypically comprising one or several bone-inducing agent (s), asdescribed above, then covers said composition with a layer of the bottomsealant, and after permitting the sealant or the composition or both topolymerize, performs the steps a-g. The nature of this type of defectsis such that as a consequence of the thinness of the subchondral bonelayer there is high probability that the lesion will extend into theunderlying cancellous bone. In such an instance, the bone-inducingcomposition or bone acellular implant is deposited into the skeletalbone and in flowable continuation into the osteochondral bone which isthen covered with the bottom sealant layer and the acellular implant isdeposited as described above.

B. Bone Defects

The true bone defects, lesions or fractures are stand alone injuries inthe skeletal bone. These types of injuries may also be convenientlytreated according to the invention with the bone-inducing composition orwith a carrier comprising such bone-inducing composition.

The carrier, in this setting, corresponds to the acellular implantutilized for treatment of bone. This bone acellular implant comprisesbone-inducing agents.

The treatment of the skeletal bone injuries comprises depositing of thebone-inducing composition into the lesion or fracture during thesurgery. Typically, the bone-inducing composition will be administereddirectly into the lesion or fracture as a powder or a solution, such asan adhesive or polymerizable solution, or the composition will beincorporated into a bone-inducing carrier or porous matrix as describedabove. The bone lesion may or may not then be covered with the topsealant or any other surface to contain the composition within thelesion.

In the preferred embodiment, the demineralized bone powder is used as apowder or in solution wherein said powder is dissolved in the collagen,hydrogel or some other adhesive solution which has no bone formingeffect. The bone-inducing composition is added in amount which willcompletely fill the lesion or fracture.

IX. Treatment of Human Osteoarthritic Cartilage

Articular cartilage is a unique tissue with no vascular, nerve, orlymphatic supply. The lack of vascular and lymphatic circulation may beone of the reasons why articular cartilage has such a poor intrinsiccapacity to heal, except for formation of fibrous or fibrocartilaginoustissue. Unique mechanical functions of articular cartilage are neverreestablished spontaneously after a significant injury, age wear ordisease, such as osteoarthritis (OA).

Currently, the only available treatment of severe osteoarthritis of theknee is a total knee replacement in elderly patients. In young andmiddle aged patients, however, this is not an optimal treatment.

Although the current invention is more practicable for treatment ofinjuries in young individuals who naturally possess sufficient levels ofextracellular matrix building enzymes, growth factors, and othermediators, the method may be advantageously modified to also providetreatment for older population.

For treatment of elderly patients or for treatment of larger lesions,the acellular matrix implant is incorporated, before implantation, withone or more metalloproteinases, mediators, enzymes and proteins and/orwith drugs stimulating endogenous production of these factors andmediators. These factors, as described above, stimulate and promotechondrocytes activation, migration and extracellular matrix secretion.The method of the invention thus is also suitable for treatment of thecartilage defects in older generation. It is expected, however, thatsuch treatment will require longer period of treatment.

In osteoarthritis, or in age worn out cartilage, disruption of thestructural integrity of the matrix by the degeneration of individualmatrix proteins leads to reduced mechanical properties and impairedfunction. Consequently, the current invention reverses this process byproviding a means for rebuilding the diseased osteoarthritic or worncartilage with the new healthy hyaline cartilage.

X. In Vivo Studies in Swine of the Weight-Bearing Region of the Knee

The method according to the invention was tested and confirmed in invivo studies in swine.

The studies, described below, were designed to evaluate feasibility ofthe porcine acellular matrix implant by detecting chondrocyte activationand induction of chondrocyte migration on the surrounding cartilage,generation of newly synthesized hyaline cartilage within the lesion andformation of superficial cartilage layer.

Studies involved the creation of defects in the weight-bearing region ofthe femoral medial condyle of the knee joint, implantation of theacellular matrix into the defect, depositing bottom and top sealants,detection of growth of a superficial cartilage layer after two weeksfollowing the defect creation, detection of chondrocyte morphology,detection of pannus invasion and presence of fibrocartilage, detectionof presence or absence of S-GAG secretion, histochemical evaluation ofpresence or absence of sealants.

Gross anatomy of the empty defect creation and acellular matriximplantation at day zero is shown in FIGS. 3 and 4. Formation of thehealthy hyaline cartilage and generation of the superficial cartilagelayer in defects treated with the acellular matrix implant and thefibrocartilage pannus invasion in control defects at seven monthfollowing the defect creation are seen in FIGS. 5-12.

FIG. 3 shows two empty defects sites A and B at a time of the defectcreation (time zero). FIG. 4 shows two defects created at time zeroimplanted with the acellular matrix implants at sites A and B.

FIGS. 5 and 6 show arthroscopic evaluation two weeks after defectcreation in the control group (FIG. 5) and in the experimental groupimplanted with the acellular matrix (FIG. 6). Histological grading isseen in FIG. 7 and histological evaluation, in two magnifications, isseen in FIGS. 8 and 9 for the control animals and in FIGS. 10 and 11 forthe experimental group treated with the acellular implant. Degradationof the sealant from the cartilage lesion is seen in FIGS. 12A-12C. Oneexample of full thickness defect at femoral condyle of mini-pig is seenin FIG. 13.

Schematic representation of the femoral articular surface, defectcreation and implant implantation sites within said defect is shown inFIG. 1D. FIG. 1D shows two defects A and B created in the femoral medialcondyle on the medial side of the femoral articular surface. The defectshave sizes of 4 mm in diameter and 1-1.5 mm in depth. The defects arecreated in the weight-bearing region.

Table 1 is a tabulation of conditions of a study design as schematicallyillustrated in FIG. 1D. TABLE 1 Study Design Number Number Group of ofNumber Animals Samples Procedure Arthroscopy Necropsy 1 8 16**Implantation 2 weeks 7 months Experi- of acellular after after mentalbiodegradable implantation implantation matrix* 2 8 16** Empty defect 2weeks 7 months Control control after after defect defect creationcreation*Matrix was secured with tissue adhesive and sutures.**Each group has two samples at weight-bearing site (site A and B, FIG.1D).

Table 1 illustrates the study design for the seven months study offeasability of the acellular matrix implant for treatment of cartilagelesions. Study involved 8 castrated male Yucatan micro-swine, 9-12months old in each of the two groups. Two defects (A and B) were createdat time zero in the knee of each animal, with a total number of 16defects. The experimental group was implanted with acellular matriximplant at a time of defect creation. In the control group, the defectwas left empty without any treatment and was used for visual,microscopical, histological and histochemical comparisons. Arthroscopywas performed at 2 weeks after implantation and defect creation.Necropsy was performed 7 months after implantation and defect creation.

The acellular matrix implant was prepared from a collagen solutionVITROGEN® (35 μL) obtained from Cohesion, Calif. The collagen gelsolution was absorbed into a collagen honeycomb sponge (5 mm in diameterand 1.5 mm in thickness) obtained from Koken Co., Japan. The combinedcollagen gel/sponge constructs seen in FIG. 2A were pre-incubated for 1hour at 37° C. to gel the collagen, followed by incubation in culturemedium with 1% penicillin and streptomycin at 37° C. at 5% CO₂. Afterabout 24 hours of polymerization, the biodegradable scaffolds weretransferred to the tissue container with pre-warmed culture medium (37°C.) for the implantation.

Arthrotomy was performed under an inhalation anesthesia. After openingknee joint capsule, two empty full-thickness defects (4 mm in diameterand about 1.5 mm in depth) were created in the femoral articularcartilage on the weight-bearing site of the medial femoral condyle ofeach animal. After creating defects, tissue sealant was placed on thebottom of the defect. Then, the pre-prepared acellular biodegradablematrix was placed over the bottom sealant within the cartilage lesion.The acellular matrix was secured with absorbable sutures (usually 4 to 6sutures) and with two non-absorbable sutures. The non-absorbable sutureswere used as a maker for arthroscopic evaluation and are visible in FIG.6. The implanted defect was then sealed with the top sealant. For thecontrols, two empty full-thickness defects were created and left intact,that is empty, without implants, or deposition of the bottom or topsealants.

FIG. 3 shows a photograph of the two empty full-thickness defects A andB (4 mm in diameter and 1-1.5 mm in depth) created in the articularcartilage on the weight-bearing site of the medial femoral condyle. Theempty defects were left intact during the whole time of the study andwere used as controls for the experimental group.

FIG. 4 is a photograph of the two full-thickness defects created in thesame way as the empty defects seen in FIG. 3. These two defects weretreated, according to the method of the invention, with the bottomsealant deposited on the bottom of the lesion. The acellular matriximplant was implanted into the lesion cavity over the bottom sealant andthe top sealant deposited the over the implanted acellular matriximplant. The implants were collagenous sponges (FIG. 2A) and had 5 mm indiameter and 1.5 mm in thickness. Both sites A and B were implanted.Each implant was secured with four absorbable sutures and twonon-absorbable sutures used as markers for future arthroscopicevaluation.

Two weeks after defect creation and acellular matrix implantation, theempty defects and implant sites were evaluated with arthroscopy.Arthroscopic evaluation after 2 weeks is seen in FIGS. 5 and 6.

FIG. 5 is an arthroscopic microphotograph of an empty defect 2 weeksafter defect creation. Arthroscopic evaluation showed that in thecontrol group, if left untreated, the lesion was invaded with synovialpannus and filled with fibrocartilage. The arthroscopic evaluationclearly shows the defect depression indicating that the defect is fullyexposed and empty although some synovial invasion have already occurred.Such synovial invasion is a first step toward formation offibrocartilage. Formation of fibrocartilage to replace the hyalinecartilage is undesirable as the fibrocartilage is qualitatively andfunctionally inferior to hyaline cartilage.

Arthroscopic evaluation of implanted sites showed that already at twoweeks time the defects are covered with the superficial cartilage layer.FIG. 6 is an arthroscopic microphotograph of the defect treated with theacellular matrix implant 2 weeks after the defect was created. The FIG.6 shows the superficial cartilage layer overgrowing the implant siteforming a smooth flat surface. The borders of the implant site arealready undefined compared to the empty defect which has a definite andvisible border, said implanted site indicating the beginning ofchondrocyte migration into the implant and secretion of extracellularmatrix in confluency with the host cartilage, all this covered with thesuperficial cartilage layer. The arthroscopic evaluation seen in FIG. 6revealed that the lesion implanted with the acellular matrix isunexposed and covered with the superficial cartilage layer completelyovergrowing the implant sites, seen as a smooth flat surface whencompared to the fully exposed and empty defects of controls, seen inFIG. 5.

At 7 months after creating the defects and implanting the acellularmatrix implants, the animals were euthanized. The implant and defectsites on the femoral articular condyle were harvested for histologicalevaluation. The tissues were fixed with 4% formaldehyde/PBS for 7 daysat 4° C. The tissues were decalcified with 10% formic acid, processed,and embedded in paraffin. Thin sections (5 μm) were stained withSafranin-O (Saf-O) and hematoxylin eosin (H-E) for histologicalevaluation.

The stained sections were evaluated blindly by means of a histologicalgrading scale seen in FIG. 7, modified from J. Bone Joint Surg. Am.,79:1452-63 (1997). Only sections from the center of the defect weregraded in order to ensure unbiased analysis and to allow comparisonamong specimens studied at different time-point. The area from thecenter of the defect was also chosen because it provided the moststringent test of healing capacity, since the least amount of cartilagehealing was found consistently in specimens taken from the middle of thedefect.

Histological scoring system used for cartilage repair evaluation is seenin Table 2. TABLE 2 Category 1. Filling of defect Score Filling ofDefect 0 None (or almost none) 1 <50% 2 >50% 3 All (or almost all) 2.Integration of repair tissue with surrounding articular cartilage ScoreIntegration 0 Gap or lack of continuity on two sides 1 Gap or lack ofcontinuity on one sides 2 Non-continuous gap or lack 3 Normal continuityand integration 3. Matrix staining with Safranin O-fast green (comparedto host cartilage) Score Matrix staining 0 None (or almost none) 1Slight 2 Moderate 3 All (or almost all) 4. Cellular morphology ScoreChondrocytes morphology 0 Mostly spindle-shape (fibrous-like) cells 1<50% of round cells with morphology of chondrocytes 2 >50% of roundcells with morphology of chondrocytes 3 Normal (mostly round cells withmorphology of chondrocytes) 5. Architecture within entire defect (notincluding margins) Score Architecture within entire defect 0 Clefts orfibrillations 1 <3 large voids 2 >3 large voids 3 Normal 6. Architectureof surface Score Architecture of surface 0 Severe fibrillation orirregularity 1 Moderate fibrillation or irregularity 2 Slightfibrillation or irregularity 3 Normal (or nearly normal) 7. Penetrationof tissue to subchondral bone area Score Penetration 0 Severepenetration 1 Moderate penetration 2 Slight penetration 3 Normal (ornearly normal)

Cumulative results of the histological grading of the repaired chondralcartilage is seen in Table 3. TABLE 3 Histological Grading of theRepaired Cartilage Acellular Matrix Empty Defect Category Group GroupFilling of defect 3.00 2.60 Integration 2.00 1.40 Matrix staining 2.332.10 Chondrocyte morphology 1.78 0.80 Architecture within entire defect2.33 0.30 Architecture of surface 2.33 1.90 Tissue penetration into 2.111.40 subchondral bone area Average total score 15.88 10.50 SD± 1.90 3.60

As seen in Table 3 the average total score for histological grading at 7months after the defect creating and treatment with the acellular matriximplant was much higher in the implant group, with the score for allindicators in the implant group being higher then in the empty defectgroup.

Histological grading of the repair tissue is shown in FIG. 7, whichgraphically illustrates results shown in Table 5. The average totalscores on the histological grading scale were significantly better(p<0.001) for the defects treated with acellular matrix implants thanfor the untreated defects.

At seven months following the defect creation, animals were sacrificed,their joints were harvested and evaluated by Safranin-O staining.Results are seen in FIGS. 8-11.

The non-implanted, empty defects A and B at 7 months after defectcreating are shown in FIGS. 8A, 8B, 9A and 9B.

FIG. 8A is a Safranin-O staining microphotograph (29× magnification) ofthe empty, non-implanted defect (D) at a control site A seven monthsafter defect creation. In higher magnification (FIG. 8B), the defectclearly shows a fibrous tissue (F) filling the defect surrounded by thehost cartilage (H) with underlying subchondral bone (SB) area (FIG. 8A).None or a very small amount of S-GAG accumulation, depicted by redcolor, was observed at the defect site. S-GAG accumulation is evidenceof the extracellular matrix formation. If there is a little or noneS-GAG present, there is no extracellular matrix generated, indicatingthe absence of migrating chondrocytes and absence of formation ofhyaline cartilage. It also indicates the presence and formation offibrocartilage within the lesion. FIG. 8B shows a 72× magnification ofthe defect site confirming a presence of fibroblasts, that is fibrouscells, indicating invasion of a fibrovascular pannus (F) from synovium.Chondrocyte morphology showed presence of mostly spindle (fibrous)cells.

FIG. 9A is a Safranin-O staining microphotograph (29× magnification) ofthe empty, non-implanted defect (D) at a site B of the control defectseven months after defect creation showing a formation of fibrous tissuefilling the defect surrounded by the host cartilage (H) with underlyingsubchondral bone (SB) area. Severe irregularity of the lesion surfacewas observed. Only very slight S-GAG accumulation, depicted by redcolor, was observed at the defect site. S-GAG accumulation is evidenceof the extracellular matrix formation.

FIG. 9B shows a 72× magnification of the defect site showing a presenceof fibroblasts indicating a fibrovascular pannus (F) invasion fromsynovium. Cell morphology observed at this site shows mostly spindlefibrous cells.

FIGS. 8A, 8B, 9A and 9B clearly show that non-implanted control defectswithout treatment with the acellular implant of the invention do notindicate a formation of the healthy hyaline cartilage which would showas S-GAG accumulation, in Safranin-O stained microphotograps seen as ared color. Rather, these microphotographs show fibrovascular pannussynovial invasion into the defect with an accumulation of spindlyfibrous cells present in the empty defect sites.

While the no-treatment of the lesion resulted in the filing of thedefect with the fibrocartilage, the implantation of the acellular matriximplant into the defect induced chondrocyte activation and migrationfrom the surrounding native cartilage and resulted in massive formationof cartilage extracellular matrix (ECM accumulation) with minimalfibrovascular pannus in the implant sites. ECM accumulation was detectedby the strong red color present at the implanted sites of experimentalanimals. Results are seen in FIGS. 10A, 10B, 11A and 11B.

FIG. 10A is a micrograph of Safranin-O staining histological evaluation(29× magnification) of the acellular matrix implant (I) implantedwithing the defect site A, seven month after defect creation andimplantation of the acellular matrix implant. FIG. 10A clearly showsinducement of cell migration from the surrounding native host cartilage(H) into the implant (I) implanted within the defect site. After sevenmonths following the implantation, hyaline-like cartilage was observedat the acellular implant site. The presence of the hyaline cartilage isindicated by the normal S-GAG accumulation, seen as a predominant redpresent in the defect site A. Superficial cartilage layer formed overthe lesion is also seen. There was minimal fibrovascular pannus in theimplant sites. Implant is surrounded by the host cartilage (H) withunderlying subchondral bone area (SB).

FIG. 10B shows a higher magnification (72×) of the implant area with redcolor indicative of S-GAG accumulation and chondrocyte morphologyshowing primarily normal mostly round cells as compared to spindlyfibrous cells observed in the non-treated control defects.

FIG. 11A is a Safranin-O staining histological evaluation (29×magnification) of the acellular matrix implant (I) implanted withing thedefect site B, seven month after implantation. FIG. 11A confirms resultsseen in FIG. 10A. It clearly shows inducement of cell migration from thesurrounding native host cartilage (H) into the implant (I) implantedwithin the defect site. At seven months after implantation, hyaline-likecartilage was observed at the acellular implant site. The presence ofthe hyaline cartilage was indicated by the normal S-GAG accumulation,seen as a predominant red color present in the defect site B.Superficial cartilage layer formed over the lesion and traces ofnon-absorbable suture are also seen. No fibrovascular pannus synovialinvasion was observed in the implant site. Implant is surrounded by thehost cartilage (H) with underlying subchondral bone area (SB). Thenon-absorbable suture indicates the original border between the hostcartilage and the implant, now almost completely obscured.

FIG. 11B shows a higher magnification (72×) of the implant area withhigh accumulation of red color indicative of S-GAG presence. Chondrocytemorphology again show primarily normal, mostly round cells confirmingresults observed at site A.

As seen in FIGS. 10A and 10B, 11A and 11B, there was clearly visibleintegration between the biodegradable acellular matrix and the hostcartilage. Such integration is not observed in FIGS. 8A and 9A where thedefect is surrounded by the normal hyaline cartilage. These figures showdifferent cell morphology at the defect sites than those at theimplantation sites seen in FIGS. 10A and 10B. Cell morphology of theempty sites shows the presence of spindly fibrous cells dissimilar tothose cells of the surrounding hyaline cartilage. Cell morphology at theimplanted sites, on the other hand, show the presence of the normal(round) cells also observed in the surrounding healthy hyalinecartilage. The implanted site thus, after seven months does not showdifference between the previously uninjured cartilage and the one formedwithin the defect following the implantation.

Additionally, the use of a top sealant deposited over the implantimplanted at a defect site had resulted in formation of the superficialcartilage layer and minimizing synovial tissue invasion at the implantsite.

A superficial cartilage layer is formed over the cartilage lesion afterthe top sealant is deposited over the lesion implanted with theacellular implant. As seen in FIG. 6, the presence of the superficialcartilage layer was already observed in two weeks after theimplantation. The top sealant which causes the superficial cartilagelayer to be formed is biodegradable and biodegrades within the time. Atthree months after the sealant deposition, remaining sealant was stillobserved at the surface area along with the superficial cartilage layer.At seven months after implantation, the top sealant was completelybiodegraded and superficial cartilage layer was formed in its place, asseen in FIGS. 10A and 11A.

In order to determine the sealant (top and bottom) degradation in vivo,articular cartilage samples implanted with an autologous chondrocyteconstruct using the scaffold matrix were stained with Safranin-O (FIGS.12A-12C). Reddish color in Safranin-O stained figures indicates S-GAGaccumulation. Purple color indicates remaining tissue adhesive withamorphous structure.

FIG. 12 thus illustrates a degradation pattern, in time, of the top andbottom sealants three months after the acellular matrix implantation. Atthat time, the superficial cartilage layer was formed over the implantand the top sealant was partially degraded. The bottom sealant was, atthree months following its deposition at the bottom of the lesion,completely degraded and removed from the lesion site.

FIG. 12A shows a surface view of the Safranin-O stained implantationsite with the superficial cartilage layer clearly visible and the smallamount of the top sealant remaining under the superficial cartilagelayer. FIG. 12B shows a side view of the Safranin-O stained implantationsite. FIG. 12C shows the bottom view of the Safranin-O stainedimplantation site where at time zero the bottom sealant was deposited.

In this test, the remaining top sealant was observed only at the surfacebetween the top of the regenerated hyaline like cartilage region andsuperficial cartilage layer (FIG. 12A). There was no indication in sideview of any remaining top or bottom sealant between the interface of theimplant site and the surrounding host cartilage (FIG. 12B). There was noremaining bottom sealant at the bottom of the lesion interfacing withthe subchondral bone region where the bottom sealant was deposited attime zero (FIG. 12C).

These results indicate that the bottom sealant is completely biodegradedand removed from the lesion site in about three month afterimplantation. At that time, there are still remnants of the top sealantvisible on the surface of the lesion where the sealant protects theacellular implant from any migration or invasion of synovium and at thesame time supports the formation of the superficial cartilage layer.With time even these remnants of the top sealant are biodegraded andremoved from the healed lesion as evidenced by a complete absence of anytop or bottom sealant at the defect site.

A reason why the top sealant is still present at three months time isthat, compared to the surface area, the side and bottom of the acellularimplant site are more active regions for cell migration which isimportant for cell integration and formation of hyaline cartilage. Inthese regions, the sealant was completely degraded within 3 months. Thisphenomenon occurred and was observed in both the cellular and acellularmatrix implantation in vivo. Cellular implant is described in copendingapplication Ser. No. 10/625,245 filed on Jul. 22, 2003.

In order to confirm that the surgical technique used for creation of thecartilage defects in control and experimental animals is distinguishedfrom the microfracture technique which penetrates the subchondral bonearea, an image of full thickness defect at femoral condyle of mini-pigwas created and is shown, with 72× magnification, in FIG. 13. FIG. 13shows a paraffin embedded and Safranin-O stained reference tissue of thecreated full thickness defect. The defect was created of non-treatedarticular cartilage and bone from the femoral condyle surrounded by thehost cartilage and underlying subchondral bone area. The remainingcalcified cartilage area is seen in the area above the subchondral bone.This tissue was utilized in all studies as a reference tissue used forhistological evaluation.

The results described above show that implantation of the biodegradableacellular matrix implant into the cartilage lesion according to theinvention induces chondrocyte migration from surrounding nativecartilage and formation of an extracellular matrix and leads tosynthesis of a new hyaline cartilage with minimal synovial invasion offibrovascular pannus at the implant sites.

Synthesis of the new hyaline cartilage was measured by the extracellularmatrix accumulation expressed as accumulation of S-GAG. Also observedwas a cell integration between the biodegradable acellular implant andthe host cartilage. The use of a bottom and top sealants and suturesprimarily to secure the implant within the defect suggest that thesecould have a secondary effect of minimizing synovial tissue invasion atthe implant site. On the other hand, the results described above andillustrated by the figures clearly show that the intact nontreatedcontrol defects result in synovial invasion of the defect withfibrovascular pannus.

The acellular matrix implant most suitable for practicing the inventioncomprises a porous honeycomb sponge of Type I atelocollagen filled witha thermoreversing hydrogel of Type I collagen sandwiched between abottom layer and a top layer of the sealant. The type I collagen cellwalls of the porous honeycomb add further strength to the sealingcapacity of the sealant by adding to the collagen-PEG chemicalinteraction analogously to the reaction of metal reinforcing bar toconcrete.

The acellular implant itself is fully biodegradable in time. During thattime the following conditions are observed in sexually mature but notfully epiphysealy-fused mini-swine. It is observed that in a 2 mm lesionof the femoral condyle covered with the top sealant, a superficialcartilage layer extending from the edge of the healthy cartilage regionperipheral to the acellular implant proceeds to overgrow the lesion andthe sealant layer. Additionally, chondrocyte migration into theacellular implant and production of the new hyaline cartilage matrixthat eventually fills and replaces the implant is observed. This newcartilage matrix is or closely resembles hyaline cartilage as measuredby sulfated glycoaminoglycan content and histological appearance. Thesource of these migrating chondrocytes are likely to be both theperipheral deeper layers of healthy chondrocytes peripheral to theacellular implant, and also the overgrown superficial cartilage layer,since it is shown that this layer is the source of differentiatedchondrocytes capable of producing hyaline cartilage. Eventuallyhyaline-like cartilage is found to fill the implant while at the sametime the implanted acellular matrix is gradually biodegraded.

In the current methodological arrangement, the top and bottom sealantsis intended to prevent debris from subchondral space to enter theimplant (bottom sealant) and to sequester the implant within a lesionspace (top sealant). The acellular matrix implant sequestered within thelesion permits chondrocytes from the surrounding healthy cartilage tomigrate and enter the matrix. Naturally applied hydrostatic pressureduring a normal physical activity promotes chondrogenesis leading to aformation of true hyaline cartilage and to a healing of the lesion.

Results of studies described above confirm that the damaged, injured,diseased or aged cartilage may be repaired by using acellular implantsprepared according to the invention and that the acellular matriximplant of the invention induces cell migration from surrounding healthyhost cartilage and its implantation induces the inward growth of thesuperficial cartilage membrane from the healthy tissue on the periphery.This membrane, superficial cartilage layer, protects the implant withinthe lesion from any synovial invasion. Once the implant is properlyimplanted within the lesion, the natural physicochemical factors, suchas intermittent hydrostatic pressure, low oxygen tension and growthfactors induce the cartilage recovery.

The advantages of the acellular matrix implant system are multiple.There is no need for biopsy and cell harvesting, no need to coverperiosteum over the lesion, no damage to healthy tissue, the second andthird surgery is eliminated resulting in faster recovery and eliminationof waiting periods for the next surgery.

Advantages listed above are similarly attached to treatments ofsubchondral or bone lesions.

EXAMPLE 1 Preparation of Acellular Collagenous Implants

This example illustrates preparation of the acellular matrix implant.

300 grams of a 1% aqueous atelocollagen solution (VITROGEN®), maintainedat pH 3.0, is poured into a 10×20 cm tray. This tray is then placed in a5 liter container. A 50 ml open container containing 30 ml of a 3%aqueous ammonia solution is then placed next to the tray, in the 5 literchamber, containing 300 grams of said 1% aqueous solution ofatelocollagen. The 5 liter container containing the open trays ofatelocollagen and ammonia is then sealed and left to stand at roomtemperature for 12 hours. During this period the ammonia gas, releasedfrom the open container of aqueous ammonia and confined within thesealed 5 liter container, is reacted with the aqueous atelocollagenresulting in gelling said aqueous solution of atelocollagen.

The collagenous gel is then washed with water overnight and,subsequently, freeze-dried to yield a sponge like matrix. This freezedried matrix is then cut into squares, sterilized, and stored under asterile wrap.

Alternatively, the support matrix may be prepared as follows.

A porous collagen matrix, having a thickness of about 4 mm to 10 mm, ishydrated using a humidity-controlled chamber, with a relative humidityof 80% at 25° C., for 60 minutes. The collagen material is compressedbetween two Teflon sheets to a thickness of less than 0.2 mm. Thecompressed material is then cross-linked in a solution of 0.5%formaldehyde, 1% sodium bicarbonate at pH 8 for 60 minutes. Thecross-linked membrane is then rinsed thoroughly with water, andfreeze-dried for about 48 hours. The dense collagen barrier has an innerimplantation of densely packed fibers that are intertwined into amulti-layer structure.

In alternative, the integration layer is prepared from collagen-baseddispersions or solutions that are air dried into sheet form. Drying isperformed at temperatures ranging from approximately 4 to 40° C. for aperiod of time of about 7 to 48 hours.

For histological evaluation, 4% paraformaldehyde-fixed, paraffinsections were stained with Safranin-O (Saf-O) and Type II collagenantibody.

For biochemical analysis, seeded sponges were digested in papain at 60°C. for 18 hours and DNA content was measured using the Hoechst 33258 dyemethod. Sulfated glycosaminoglycan (S-GAG) accumulation was measuredusing a modified dimethylmethylene blue (DMB) microassay.

EXAMPLE 2 Biochemical and Histological Assays

This example describes assays used for biochemical and histologicalstudies.

For biochemical (DMB) assay, the implant taken from the animal aftercertain time following the implantation, transferred to microcentrifugetubes and digested in 300 μl of papain (125 μg/ml in 0.1 M sodiumphosphate, 5 mM disodium EDTA, and 5 mM L-cysteine-HCl) for 18 hours at600C. S-GAG production in the implant is measured using a modifieddimethylene blue (DMB) microassay with shark chondroitin sulfate as acontrol according to Connective Tissue Research, 9: 247-248 (1982).

DNA content is determined by Hoechst 33258 dye method according to Anal.Biochem., 174:168-176 (1988).

For histological assay, the remaining implants from each group werefixed in 4% paraformaldehyde. The implants were processed and embeddedin paraffin. 10 μm sections were cut on a microtome and stained withSafranin-O (Saf O).

For immunohistochemistry, the samples are contacted withdiaminobenzidine (DAB). The DAB is a color substrate showing brown colorwhen the reaction is positive.

EXAMPLE 3 Evaluation of Integration of Acellular Matrix Implant in aSwine Model

This example describe the procedure and results of study performed forevaluation of integration of porcine in a swine model.

An open arthrotomy of the right knee joint was performed on all animals,and a biopsy of the cartilage was obtained.

A defect was created in the medial femoral condyle of the pig's rightknee. This defect (control) was not implanted with an acellular matriximplant but was left intact. Following surgery, the joint wasimmobilized with an external fixation implant for a period of about twoweeks. Two weeks after the arthrotomy on the right knee was performed,an open arthrotomy was performed on the left knee and defects werecreated in this medial femoral condyle. The acellular matrix implant wasimplanted within the defect (s) in this knee which was similarlyimmobilized. The operated sites were subsequently viewed via arthroscopytwo weeks after implantation or defect creation and thereafter atmonthly intervals.

Animals were euthanized and the joints harvested and prepared forhistological examination approximately 7 months after acellular matriximplant implantation. The implanted sites were prepared and examinedhistological.

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 30. An acellular matrix implant for implantation into aninjured, damaged, diseased or aged cartilage, said implant comprising acollagenous construct a construct prepared from a polymer of an aromaticorganic acid, or a construct prepared from a thermo-reversible hydrogel,sol-gel or gel material.
 31. The implant of claim 30 wherein saidconstruct further comprises a compound selected from the groupconsisting of a mediator, growth factor, enzyme, protein, peptide and adrug able to enhance endogenous production of said mediator, factor,enzyme, protein or peptide.
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 38. The implant of claim 31wherein said enzyme, protein or peptide is selected from the groupsconsisting of a matrix remodeling enzyme, a matrix metalloproteinase,aggrecanase and cathepsin.
 39. The implant of claim 30 wherein saidconstruct is prepared from a material selected from the group consistingof a Type I collagen, a Type II collagen, a Type IV collagen, acell-contracted collagen containing proteoglycan, a cell-contractedcollagen containing glycosaminoglycan, a cell-contracted collagencontaining glycoprotein, a polymer of an aromatic organic acid, gelatin,agarose, hyaluronin, fibronectin, laminin, a bioactive peptide growthfactor, a cytokine, elastin, fibrin, a polymer of an aromatic organicacid or a copolymer thereof, a synthetic polymeric fiber made ofpolylactic acid, a synthetic polymeric fiber made of polyglycolic acid,a polycaprolactone, a polyamino acid, a polypeptide gel, a polymericthermo-reversible gelation hydrogel (TRGH), a copolymer thereof and acombination thereof.
 40. The implant of claim 30 wherein said constructis a biodegradable collagenous sponge, collagenous porous sponge,collagenous honeycomb sponge or a collagenous scaffold.
 41. The implantof claim 40 wherein said sponge or scaffold is prepared from Type Icollagen.
 42. The implant of claim 41 wherein said sponge or scaffold isa porous construct having a size from 1 to 5 mm in diameter, about 1.5mm in thickness and pores sizes from about 200 to about 400 μm.
 43. Theimplant of claim 42 wherein said sponge or scaffold is incorporated witha thermo-reversible gelation hydrogel as a sol at temperatures betweenabout 5 to about 30° C., wherein at the body temperature saidthermo-reversible gelation hydrogel converts from a fluidic sol into asolid gel.
 44. The implant of claim 39 wherein said construct isprepared from the thermo-reversible gelation hydrogel.
 45. The implantof claim 44 wherein said thermo-reversible gelation hydrogel (TRGH)construct is deposited into a lesion cavity of the injured, damaged,diseased or aged cartilage cavity as a fluidic sol at a temperaturebetween about 5 to about 30° C., wherein within said cavity and at thebody temperature said thermo-reversible gelation hydrogel converts fromthe fluidic sol into a solid gel.
 46. The implant of claim 39 whereinsaid construct is prepared from the polymer of the aromatic organicacid.
 47. The implant of claim 30 wherein said construct isbiocompatible, biodegradable, hydrophilic and has a neutral charge. 48.A collagenous acellular matrix implant for implantation into an injured,damaged, diseased or aged cartilage lesion, said implant prepared from acollagenous material fabricated as a sponge, porous sponge, scaffold,porous scaffold, honeycomb, porous honeycomb, honeycomb scaffold,lattice or porous lattice.
 49. The implant of claim 48 that isbiocompatible, biodegradable and hydrophilic.
 50. The implant of claim49 prepared from a material selected from the group consisting of a TypeI collagen, a Type II collagen, a Type IV collagen, a cell-contractedcollagen containing proteoglycan or a cell-contracted collagencontaining glycosaminoglycan or glycoprotein.
 51. The implant of claim50 wherein said implant is a porous construct having a size from 1 to 5mm in diameter, about 1.5 mm in thickness and pores sizes from about 200to about 400 μm.
 52. The implant of claim 51 wherein the size of theimplant is adjusted to the size of the cartilage lesion.
 53. The implantof claim 52 wherein said implant is incorporated with athermo-reversible gelation hydrogel as a sol at temperatures betweenabout 5 to about 30° C., wherein at the body temperature saidthermo-reversible gelation hydrogel converts from a fluidic sol into asolid gel.